Compositions and methods to selectively control invasive species

ABSTRACT

Methods and compositions for controlling the growth of an invasive species by application of a composition comprised of a natural pesticide derived from a species to the invasive species, especially endocides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/889,184, filed Nov. 5, 2015, which is a national phaseapplication under 35 U.S.C. § 371 of International Application No.PCT/US2014/036837, which claims the benefit of priority of U.S.Provisional Application No. 61/819,713, filed May 6, 2013. The entirecontents of each of the above-referenced disclosures are herebyincorporated by reference.

BACKGROUND OF THE INVENTION A. Field of the Invention

The present invention relates generally to the fields of Biology andChemistry. More particularly, it concerns compositions and methods forcontrolling the growth of invasive species by endocides.

B. Description of Related Art

Invasive species are major threats to agriculture, natural environments,and public health (Ascunce et al. 2011). The estimated annual damagefrom invasive species worldwide totals more than $1.4 trillion—fivepercent of the global economy (The Nature Conservancy, 2013). Theprinciple of the current management strategies of invasive species inthe world is mainly restricted to eliminate or eradicate the species byusing synthetic herbicide, biological or mechanical applications. Suchcontrol measures of invasive species are difficult and often veryexpensive (Beck et al. 2008). Controlling invasive species and theassociated economic and environmental damages amounts to more than $138billion per year (Pimentel et al. 1999). More than $500 million is spenton residential exotic weed control and an additional $1 billion isinvested in non-indigenous weed control on golf courses (Pimentel et al.1999). In addition to huge expenditures, effectiveness decreases as morespecies develop resistance to herbicides and public environmentalconcerns regarding chemical and biological applications increase, itrequires a novel philosophy to successfully control the noxious invasivespecies.

Five of the most aggressive and noxious species in the southeasternUnited States are representative of different groups of invasivespecies. They include one fern species: giant Salvinia (Salvinia molestaD. S. Mitchell) (family Salviniaceae) from the phyllum Pteridophyta ofthe kingdom Plantae, two flowering plant species: Brazilian pepper tree(Schinus terebinthifolius Raddi) (family Anacardiaceae) from the orderSapindales and Chinese tallow tree (Triadica sebifera (L.) Small)(family Eurphorbiaceae) from the order Malpighiales of the phyllumFlowering Plants of the kingdom Plantae, and two insect species: the redimported fire ant (Solenopsis invicta Buren) (family Formicidae) fromthe superorder Endopterygota and the subterranean termite(Reticulitermes flavipes (Kollar)) (family Rhinotermitidae) ofsuperorder Exopterygota of the phylum Arthropoda of the kingdomAnimalia. Of these species, giant Salvinia is an aquatic species and theothers are terrestrial species.

Salvinia Séguier is a genus of floating ferns belonging to the familySalviniaceae Reichenbach and has 10-14 species in the world,particularly in the tropics. Salvinia molesta D. S. Mitchell, known asgiant Salvinia, water fern, or kariba weed, is native to Brazil. Since1939, it has invaded lake and river systems in warm climates in theworld (Room et al. 1990). At present, giant Salvinia is one of the mostwidespread and environmentally, economically and socially destructiveinvasive plant species (Schooler et al. 2011). In addition, giantSalvinia provides habitat for snails that are intermediate hosts forSchistosoma sp. which parasitize the human intestinal and urinarytracts. The parasitic disease schistosomiasis is also known as snailfever, bilharzia, or bilharziosis, is the second most socioeconomicallydevastating parasitic disease after malaria.

Giant Salvinia is able to double in number and biomass in less thanthree days under optimal condition and forms dense mats over stillwaters (Barrett 1989). The plant can regenerate vegetatively even aftersevere damage or drying for days (Finlayson 1984; Room and Thomas 1986).The explosive growth of S. molesta adversely affects the naturalecological system of the infested region, and it also causesconsiderable economic damage and sanitation problems. Dense mats of S.molesta reduce dissolved oxygen levels and block all sunlight frompenetrating the infested water body. Thus, macrophytes and microscopicalgae that form the base of the food chain may die off (Room et al.1990). The creatures that feed on these may die, too, and so on up thefood chain. This pest threatens cultivated aquatic crops, and it canclog irrigation and drinking water lines and foul hydroelectric plants.Salvinia-infested waters cannot be used for boating or otherrecreational purposes (USDA 2000). Biological, mechanical, andherbicidal control of invasive giant Salvinia is very expensive and hasnot been successful. Since 1980, the tiny Salvinia weevil (Cyrtobagoussalviniae, Curculionidae) has been introduced into most regions wheregiant Salvinia has invaded (Julien et al. 2009). The weevil is a strictspecialist with adults feeding on Salvinia buds; the plant is highlysusceptible to the insects and thus the weevil has successfullycontrolled Salvinia for years in some regions. But recently it was foundthat the biological control is incomplete and fitful associated withstochastic flooding events and thus is unpredictable (Schooler et al.2011). Other attempts to control and eradicate S. molesta throughherbicides and mechanical means have failed to achieve their purpose andmay cause environmental backlashes due to the introduction of chemicalsor bioagents into the environment (Abbasi and Nipaney 1986).

Brazilian pepper tree (Schinus terebinthifolius Raddi) (familyAnacardiaceae) is an evergreen shrub or tree (up to 12 m in height)native to South America and was introduced to North America in the 1800sas an ornamental plant. Like poison ivy or sumac of the same family,this species may also cause dermatitis to people with sensitive skin andeven respiratory problems during its bloom period. Brazilian pepper treehas extensively infested landscapes in Florida and south Texas and itproduces a dense canopy that shades out other plants particularly nativespecies. It is reported that its aqueous extract inhibited the growthand germination of native Florida plants (Morgan and Overhlt 2005). Theinvasiveness of Brazilian pepper tree can be attributed to its high seedproduction, high germination rates and dispersal by birds and mammals.Currently, small pepper tree seedlings are controlled by digging orpulling and also by the application of herbicides.

Chinese tallow tree (Triadica sebifera (L.) Small) (familyEurphorbiaceae) is native to China and is now a very invasive species inthe southeastern United States. A mature tree may annually produce anaverage of 100,000 seeds that are spread mainly by birds and water(Jubinsky and Anderson 1996). Under the parent tree, there maybeapproximately 15 seedlings per square meter, and its relative frequencyof tallow seedlings was greater than that of all species except forsweetgum (Liquidambar styraciflua L.). Various measures, includingmanual and mechanical, environmental/cultural, chemical, and biologicalmethods have been used to control this invasive species.

Since its introduction from its native South American range in the1930s, the red imported fire ant (Solenopsis invicta Buren) (familyFormicidae) has rapidly widespread throughout the southern UnitedStates. It has also recently invaded other regions of the world,including the Caribbean, Mexico, Australia, New Zealand, Malaysia,Singapore, and China (Chen et al. 2009; Ascunce et al. 2011). Unlikeother insects in Hymenoptera, the red imported fire ant contains a smallfraction of proteins in its venom. About 95% of the fire ant venomconsists of alkaloids (primarily 2-methyl-6-alkyl or alkenylpiperidines), which are responsible for the immediate hive formation andthe development of the sterile pustule at the sting site (Chen et al.2009; Hoffman 2010). The pest detrimentally impacts human health,livestock, wildlife, crops, machinery, and electrical equipment(Morrison et al. 2004). The estimated cost of control, medicaltreatment, and damage to property by fire ant in the United States aloneis more than $6 billion annually (Ascunce et al. 2011). An effectivemeasure for control is needed for both ecological and economic reasons.

Eastern subterranean termite (Reticulitermes flavipes (Kollar)) (familyRhinotermitidae) is the most common termite found in North America. Thisnative termite is one of the most economically important wood destroyingpests in the United States and it causes billions of dollars in homedamage each year. Currently, termite management includes reducing thepotential for termite infestation, preventing entry, and chemicalcontrol.

The use of defensive chemicals is commonly reported throughout the plantand animal kingdoms. It is known that exceptional success of someinvasive species is because they produce toxic secondary metabolites andhave negative allelopathic effects on neighboring plants or to protectthemselves from microbial attacks and insect/animal herbivory (Quintanaet al. 2008; Paudel 2009). Studies on defensive or allelopathic effectsof chemicals are mainly to inter-species. Autotoxicity (autointoxicationor intraspecific allelopathy in plants) has not been well investigatedalthough it has been hypothesized to exist since the 1970s (McKey 1974;Fowden and Lea 1979; Li et al. 2010). The term was used in plants when aspecies releases toxic substances into the environment that inhibit ordelay germination and growth of the same species (Chou and Lin 1976;Singh et al. 1999). There are few autotoxicity reports in crops andweeds and resulting soil sickness and replanting problems (Singh et al.1999). These reported autotoxic chemicals had broad-spectrumallelopathic effects and were more effectively inhibited innon-closely-related species in other genera, families or orders than inthe parent species (Abdul-Rahman and Habib 1989; Heisey 1999; Batish etal. 2002). However, many of these reports are general allelopathy ratherthan autotoxicity cases. For example, the effective concentrations ofmomilactone A and B from rice (Oryza sativa L.) (family Poaceae) toinhibit the seedlings of rice cultivars were 100 and 333 times,respectively of those to inhibit four species of Brassicaceae,Asteraceae, and Fabaceae and five weed grass species of Poaceae(Kato-Noguchi and Ota 2013). Leaf extracts from Leucaena leucocephala deWit. (family Fabaceae) had significant toxic effects on crop or treespecies belonging to other genera of Fabaceae or other families but werenot toxic to L. leucocephala seedlings (Chou and Kuo 1986). Minosineisolated from L. leucocephala had more potent inhibitory activity on theseedlings of five species of Brassicaceae, Asteraceae, Fabacreae, andPoaceae but did not show any inhibition on the producing plants at 100mg/L (Xuan et al. 2006). Leaf and flower extracts of ragweed (Partheniumhysterophorus L.) (family Asteraceae) inhibited the germination andgrowth of several species in Brassicaceae, Fabaceae, and Poaceaealthough they showed autotoxic effects to the producing plants (Picmanand Picman 1984; Javaid et al. 2007). Alfalfa (Medicago sativa L.)(family Fabaceae) is one of the most extensive investigated andwell-known autotoxicity species. It was reported that alfalfa extractsand its phenolics or saponins showed autotoxic effects on seedlinggrowth of alfalfa and allelopathic effects on several crop and weedspecies of Fabaceae, Poaceae, and Brassicaceae (Wyman-Simpson et al.1991; Chon et al. 2002). However, alfalfa allelopathy seems to be moresevere than autotoxicity (Hedge and Miller 1990). Such non-selectivityor low toxicity of the reported autotoxic chemicals to the parentspecies over other species limited the development of biocide toselectively control an invasive species. In fact, it has been widelybelieved that a species can avoid self-toxicity by its endogenouscytotoxic metabolites (Baldwin and Callahan 1993; Wang 1996; Gog et al.2005; Sirikantaramas et al. 2008a, b). Therefore, studies inautotoxicity have been primarily focused on avoidance of autotoxicityand detoxification mechanisms. To date there is no method or productdeveloped to use any autotoxic chemicals to selectively control theinvasive parent species.

SUMMARY OF THE INVENTION

Disclosed herein are methods and compositions for controlling the growthof a first invasive species by application of a composition comprising anatural pesticide derived from a second invasive species to the firstinvasive species. In some embodiments, the invasive species is aninvasive species with glands as the primary accumulation sites ofautotoxic chemicals. The first and second invasive species may be thesame or they may be different.

In some embodiments, provided are methods of controlling the growth ofan invasive species comprising applying a composition comprising anatural pesticide to the invasive species, wherein the natural pesticideis derived from an invasive species. Any measure of decreased growth orreproduction of the invasive species is contemplated. In someembodiments, the growth of the invasive species may be slowed or halted.In some embodiments, the invasive species may be eliminated. In someembodiments, the growth of the invasive species is halted within 1, 2,3, 4, 5, 6, 7 days, 1, 2, 3, 4, or 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 months or more. In some embodiments, the growth of theinvasive species is halted within 1 week or 1 month. In someembodiments, the growth of the invasive species is halted for at least1, 2, 3, 4, or 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months,1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more. In some embodiments, thegrowth of the invasive species is halted for at least 1 year.

In some embodiments, the natural pesticide may be an endocide. Anendocide (endogenous biocide) is a biocide derived from an endogenousbioactive agent (e.g., a secondary metabolite) that does not causeapparent poison in normal growth of the producing species but willpoison or inhibit and even eliminate the parent species when induced inproducing species. It can selectively eliminate the parent species (andpossibly its closely-related species) when externally applied. The deadtissues of the species caused by an endocide will enhance the endocidalfunction to the species. The endocide may have effects especially in thespecies with glands as accumulation sites of autotoxic chemicals. Anendocide may have effects in all or some growth stages of the species(e.g., primary, secondary, and tertiary stages) and in all or selectivetissues (vegetative or reproductive tissues).

Application of endocides at low concentrations (dosages) may be used incontrol a selected part (e.g., a specific cell(s), tissue(s), ororgan(s)) of the producing species. Application of endocides at lowconcentrations (dosages) may produce abnormal morphogenesis. In someembodiments, the endocides may induce mutations. In some embodiments,the induced mutations may be used to produce desirable genotypes. Insome embodiments, the induced biosynthesis of endogenous autotoxicchemicals by a method intended to stimulate gland development may alsoproduce abnormal morphogenesis, mutations, and desirable genotypes.

In some embodiments, the endocide is an extract from the invasivespecies to be controlled. In some embodiments, the endocide is afraction of extract from the invasive species to be controlled. In someembodiments, the endocide is dry matter from the invasive species to becontrolled. In some embodiments, the endocide is fresh matter from theinvasive species to be controlled. The natural pesticide “derived from aspecies” may be any composition or compound originally obtained from aspecies, even if further modified. It also encompasses syntheticcompounds that are equivalent to the compounds derived from the speciesor derivatives thereof.

In some embodiments, the natural pesticide is derived from a second,distinct species of invasive species. In some embodiments, the naturalpesticide is a compound isolated from the second invasive species.

The natural pesticides may be any appropriate material. In someembodiments, the natural pesticide is the dry or fresh matter. In someembodiments, the natural pesticide is an extract or fraction thereof. Insome embodiments, the extract is an aqueous or organic extract orfraction thereof. In some embodiments, the extract is a dry orsolubilized extract. In some embodiments, the natural pesticide is drymatter. In some embodiments, the intact dry matter is used forproduction of the natural pesticides. In some embodiments, the drymatter is ground or processed. In some embodiments, the naturalpesticide is a concentrated extract or fraction from dry matter. In someembodiments, the natural pesticide is fresh matter. In some embodiments,the intact fresh matter is used for production of the naturalpesticides. In some embodiments, the fresh matter is chopped or blended.In some embodiments, the natural pesticide is an extract (juice)obtained directly from chopped, blended, or/and expressed fresh matter.In some embodiments, the natural pesticide is a concentrated extractjuice from chopped, blended, or/and expressed fresh matter. In someembodiments, the natural pesticide is a diluted extract juice fromchopped, blended, or/and expressed fresh matter. In some embodiments,the natural pesticide is a compound isolated from the species ofinvasive species to be controlled. In some embodiments, the naturalpesticide is a compound isolated from a second species of invasivespecies.

In some embodiments, the natural pesticide is ground dried matter in awater-penetrating bag. In some embodiments, the bag material is fabric,nylon, plastic, compostable, or biodegradable. In some embodiments, thenatural pesticide is ground dried species matter in compressed form(e.g., compressed brick or pie (“tea brick”)) without a bag. The teabrick can be prepared under appropriate moisture (7-15%), temperature(50-90° C.), and pressure (250-500 kPa). It may float around or on thesurface of the invasive species to be controlled and may break down anddissolve in the water within a few minutes after contact with water ismade. It may mix with local soils to prepare a sinkable tea brick whichcan contact water quickly and be more effective.

In some embodiments, production of extract from the dried species mattermay include processes of fresh matter collection of harvest, drying,grounding, extraction, filtration, concentrating, formula preparation,and spray application. In some embodiments, extraction is aqueous ororganic solvent extraction, distillation, infusion, decoction, orpercolation.

In some embodiments, production of extract (juice) from fresh speciesmatter may include processes of fresh matter collection or harvest,shredding or pulping, extraction, filtration, formula preparation, andspray application. In some embodiments, extraction an expression method(e.g., hydraulic pressing, screw pressing, expeller pressing, spongeexpression, abrasion, and Écuelle à piquer).

The natural pesticide may have selective activity against the invasivespecies to be controlled over other species. The selectivity is moresignificant when the natural pesticide is an extract. In someembodiments, the natural pesticide may have selective activity againstthe invasive species to be controlled and its closely-related speciesover other species. In some embodiments, the closely-related species isa species within the same genus. In some embodiments, theclosely-related species is a species within the same family. In someembodiments, the closely-related species is a species within the sameorder. In some embodiments, a composition comprising the naturalpesticide can eliminate or inhibit the invasive species. In someembodiments, a composition comprising the natural pesticide caneliminate or inhibit the invasive species and its closely-relatedspecies (e.g., a species of the same order) of the invasive species. Insome embodiments, a composition comprising the natural pesticide doesnot inhibit or slightly inhibit a native species that is not in thegenus, family, or order of the invasive species. In some embodiments, acomposition comprising the natural pesticide does not inhibit orslightly inhibit an invasive species that is not in the genus, family,or order of the invasive species.

In some embodiments, the natural pesticide is a compound of formula:

wherein each R₁, R₂, R₃, R₄ is independently —H, —OH, -halogen, —NH₂,—COOH, —C(O)NH₂, —SH, —S(O)NH₂, —S(O)₂NH₂, —OCH₂OR₁₃, —OC(O)R₁₃,—OC(O)OR₁₃, —OC(O)NR₁₃, —OP(O)(OR₁₃)₂, —SR₁₃, —S(O)₂NHR₁₃,—OP(O)(OR₁₃)₂, —SOR₁₃, —S(O)₂R₁₃, —NHC(O)R₁₃, —NHSOR₁₃, NHS(O)₂R₁₃,—OPO(OR₁₄)₂, —O-arylPO(OR₁₄)₂, or —O-alkylarylPO(OR₁₄)₂, —NHR₁₃,—N(R₁₃)₂, —C(S) R₁₃, —OR₁₃,

Each R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ is independently —H, —OH,-halogen, —CN, —NH₂, —NO₂, —COOH, —C(O)NH₂, —SH, —S(O)NH₂, —S(O)₂NH₂,—C₁-C₁₀ (oxy)alkyl, —C₁-C₁₀ alkyl, —C₁-C₁₀ alkoxy, —C₁-C₁₀(hydroxyl)alkyl, —C₁-C₁₀ (amino)alkyl, —C₁-C₁₀ (halo)alkyl, —C₂-C₁₀alkenyl, —C₂-C₁₀ (aryl)alkyl, —CH₂OR₁₃, —OCH₂OR₁₃, —OC(O)R₁₃, —C(O)R₁₃,—OC(O)OR₁₃, —OC(O)NR₁₃, —C(O)O R₁₃, —C(O)NR₁₃, —OP(O)(OR₁₃)₂, —SR₁₃,—S(O)₂NHR₁₃, —C(O)NR₁₃, —OP(O)(OR₁₃)₂, —SR₁₃, —S(O₂)NHR₁₃, —SOR₁₃,—S(O)₂R₁₃, —NHC(O)R₁₃, —NHSOR₁₃, NHS(O)₂R₁₃, —OPO(OR₁₄)₂,—O-arylPO(OR₁₄)₂, or —O-alkylarylPO(OR₁₄)₂, -isocyanate, -azido, and

R₁₄ is —H, —C₁-C₁₀ alkyl, C₇-C₁₃ arylalkyl, C₁-C₁₀ aminoalkyl, C₁-C₁₀haloalkyl, C₁-C₁₀ hydroxyalkyl, C₁-C₁₀ alkoxy, -isocyanate, -azido,-imino, -thio, and R₁₃ is —H, —C₁-C₁₀ alkyl, —(C₃-C₇) cycloalkyl,—C₁-C₁₀ (halo)alkyl, -aryl, —C₂-C₁₀ alkenyl, —C₂-C₁₀, —C₂-C₁₀ alkynyl,—C₁-C₁₀ (aryl)alkyl, —C₂-C₁₀ (aryl)alkenyl, —C₂-C₁₀ (aryl)alkynyl,—C₁-C₁₀ (hydroxyl)alkyl, —C₁-C₁₀ alkoxy, —C₁-C₁₀ (amino)alkyl,-isocyanate, -azido, -imino, -thio, -alkenyloxy, -alkynyloxy, -aryloxy,-aralkoxy, -heteroaryloxy, -acyloxy, -alkoxyamino, -alkenylamino,-alkynylamino, -arylamino, -aralkylamino, -heteroarylamino,-alkylsulfonylamino, -heterocycloalkyl, -heteroaryl.

In some embodiments, the natural pesticide is a compound of formula:

wherein R₁ is OH or α-H; and R₂ is β-H or OH. In some embodiments,R₁=OH, R₂=β-H. In some embodiment, R₁=α-H, R₂=OH. In some embodiments,R₁=OH, R₂=OH. In some embodiment, R₁=α-H, R₂=β-H. These compounds may beextracted and isolated from plant material or may be chemicallysynthesized.

The invasive species may be any appropriate invasive plant. In someembodiments, the invasive plant is an aquatic plant. In someembodiments, the invasive aquatic plant is Salvinia adnata Desvaux, S.auriculata Aublet, S. biloba Raddi, S. cucullata Roxb. ex Bory, S.cyathiformis Maxon, S. hastate Desvaux, S. herzogii de la Sota, S.martynii Kopp, S. minima Baker, S. molesta, S. natans (L.) Allioni, S.nymphellula Desvaux, S. oblongifolia Martius, S. radula Baker, S.rotundifolia Willd., S. sprucei Kuhn, Azolla caroliniana Willd. (familyAzollaceae, sometimes the genus Azolla Lam. was placed in the familySalviniace), A. circinata Oltz & Hall, A. cristata Kaulf, A.filiculoides Lam., A. japonica Franch. & Sav., A mexicana C. Presl, A.microphylla Kaulf, A. nilotica Decne. ex Mett., A. pinnata R. Br., A.rubra R. Br., Lemna minuta Kunth (family Araceae), Alternantheraphiloxeroides (Matt.) Griseb. (family Amaranthaceae), Colocasiaesculenta (L.) Schott (family Araceae), Eichharnia crassipes (Martius)Solms (family Pontederiaceae), Hydrilla verticillata (L. f.) Royle(family Hydrocharitaceae), Hygrophila polysperma Anderson (familyAcanthaceae), Hymenachne amplexicaulis (Rudge) Nees (family Poaceae),Ipomoea aquatica Forssk (family Convolvulaceae), Myriophyllum spicatumL. (family Haloragaceae), Panicum repens L. (family Poaceae), Pennisetumpurpureum Schumach (family Poaceae), Pistia stratiotes L. (familyAraceae), or Urochloa mutica (Forssk.) T. Q. Nguyen (family Poaceae). Inparticular embodiments, the invasive plant is S. molesta, S. minima, A.caroliniana, L. minuta, E. crassipes, or P. stratiotes.

The invasive species may be any appropriate invasive plant. In someembodiments, the invasive plant is a terrestrial plant. In someembodiments, the invasive plant is Acacia mearnsii De Wild (familyFabaceae), Acroptilon repens (L.) DC. (family Asteraceae), Ailanthusaltissima (P. Mill.) Swingle (family Simaroubaceae), Akebia quinata(Houtt.) Decne. (family Lardizabalaceae), Albizia julibrissin Durazz.(family Fabaceae), Alliaria petiolata (M. Bieb.) Cavara & Grande (familyBrassicaceae), Ampelopsis brevipedunculata (Maxim.) Trautv. (familyVitaceae), Ardisia crenata Sims (family Myrsinaceae), Arundo donax L.family Poaceae), Berberis thunbergii DC. (family Berberidaceae), Bromustectorum L. (family Poaceae), Broussonetia papyrifera (L.) L'Hér. exVent. (family Moraceae), Cardaria pubescens (C. A. Mey.) Jarmolenko(family Brassicaceae), Carduus nutans L. (family Asteraceae), Celastrusorbiculatus Thunb. (family Celastraceae), Centaurea stoebe L. spp.micranthos (Gugler) Hayek (family Asteraceae), Centaurea stoebe L. spp.micranthos (Gugler) Hayek, C. solstitialis L., C. diffusa Lam., C.calcitrapa L. (family Asteraceae), Cecropia peltata L. (familyUrticaceae), Chromolaena odorata (L.) King & H. E. Robins (familyAsteraceae), Cinchona pubescens Vahl (family Rubiaceae), Eichharniacrassipes (Martius) Solms (family Pontederiaceae), Cinnamomum camphora(L.) J. Presl (family Lauraceae), Cirsium arvense (L.) Scop. (familyAsteraceae), Clidemia hirta (L.) D. Don (family Melastomataceae),Cynoglossum officinale L. (family Boraginaceae), Cytisus scoparius (L.)Link (family Fabaceae), Dioscorea alata L., D. bulbifera L., or D.oppositifolia L. (family Dioscoreaceae), Dipsacus fullonum L. (familyDipsacaceae), Elaeagnus angustifolia L., E. pungens Thunb., or E.umbellata Thunb. (family Elaeagnaceae), Elymus repens (L.) Gould (familyPoaceae), Eragrostis curvula (Schrad.) Nees family Poaceae), Euonymusalatus (Thunb.) Sieb. or E. fortunei (Tursz.) Hand.-Maz. (familyCelastraceae), Euphorbia esula L. (family Euphorbiaceae), Firmianasimplex (L.) W. Wright (family Sterculiaceae), Frangula alnus Mill.(family Rhamnaceae), Hedera colchica (K. Koch) K. Koch, H. helix L. H.hibernica (G. Kirchn.) Bean (family Araliaceae), Hedychium gardnerianumSheppard ex Ker Gawl (family Zingiberaceae), Heracleum mantegazzianumSommier & Levier (family Apiaceae), Hiptage benghalensis (L.) Kurz(family Malpighiaceae), Imperata cykindrica (L.) P. Beauv. (familyPoaceae), Lespedeza bicolor Turcz. L. cuneata (Dum. Cours.) G. Don, or.L. thunbergii (DC.) Nakai (family Fabaceae), Ligustrum japonicum Thunb.,L. lucidum Ait., L. obtusifolium Sieb. & Zucc., L. ovalifolium Hassk.,L. sinense Lour., or L. vulgare L. (family Oleaceae), Linaria dalmatica(L.) Mill., L. vulgaris Mill. (family Scrophulariaceae), Liriope muscari(Decne.) L. H. Bailey (family Liliaceae), Lonicera x bella Zabel, L.fragantissima Lindl. & Paxon, L. japonica Thunb., L. maackii (Rupr.)Herder, L. morrowii A. Gray, L. tatarica L. (family Caprifoliaceae),Lygodium japonicum (Thunb.) Sw. and L. microphyllum (Cay.) R. Br.(family Lygodiaceae), Leucaena leucocephala (Lam.) de Wit. (familyFabaceae), Lythrum salicaria L. (family Lythraceae), Mahonia bealei(Fortune) Carrière (family Berberidaceae), Melia azedarach L. (familyMeliaceae), Microstegium vimineum (Trin.) A. Camus family Poaceae),Mimosa pigra L. (family Fabaceae), Miscanthus sinensis Andersson familyPoaceae), Morella faya Aiton (family Myricaceae), Nandina domesticaThunb. (family Berberidaceae), Onopordum acanthium L. (familyAsteraceae), Opuntia stricta (Haw.) Haw (family Cactaceae), Paulowniatomentosa (Thnb.) Sieb. & Zucc. ex Zucc. (family Paulowniaceae),Phyllostachys aurea Carr. ex A.& C. Rivière (family Poaceae), Pinuspinaster Aiton (family Pinaceae), Polygonum cuspidatum Siebold & Zucc(family Polygonaceae), Poncirus trifoliata (L.) Raf (family Rutaceae),Prosopis glandulosa Torr. (family Fabaceae), Psidium cattleianum Sabine(family Myrtaceae), Pueraria montana (Lour.) Merr. (synonyms: P. lobata(Willd.) Ohwi, P. montana var. lobata (Willd.) Maesen & S. Almeida),family Fabaceae), Pyrus calleryana Decne. (family Rosaceae), Ranunculusficaria L. (family Ranunculaceae), Rhamnus cathartica L. (Rhamnaceae),Rosa bracteata J. C. Wendl., R. laevigata, or R. multiflora Thunb., exMurr. (family Rosaceae), Rubus ellipticus Sm. (family Rosaceae),Schedonorus phoenix (Scop.) Holub. (family Poaceae), Schinusterebinthifolius Raddi (family Anacardiaceae), Securigera varia (L.)Lassen (family Fabaceae), Solanium viarum Dunal (family Solanaceae),Solidago canadensis L. (family Asteraceae), Sorhum halepense (L.) Pers.family Poaceae), Spathodea campanulata P. Beauv. (family Bignoniaceae),Sphagneticola trilobata L. (family Asteraceae), Spiraea japonica L. f.(family Rosaceae), Tamarix ramosissima Ledeb. (family Tamaricaceae),Striga asiatica (L.) Kuntze (family Scrophulariaceae), Taeniatherumcaput-medusae (L.) Nevski (family Poaceae), Triadica sebifera (L.) Small(synonym: Sepium sebiferum (L.) Roxb., family Euphorbiaceae), Tussiliagofarfara L. (family Asteraceae), Vernicia fordii (Hemsl.) Airy-Shaw(family Euphorbiaceae), Vinca major L. or V. minor L. (familyApocynaceae), Vitex rotundifolia L. f. (family Lamiaceae), Ulexeuropaeus L. (family Fabaceae), Wisteria floribunda (Willd.) DC. or W.sinensis (Sims) DC. (family Fabaceae).

The invasive species may be any appropriate invasive insect. In someembodiments, the invasive insect is an ant species. In some embodiments,the invasive species Acrolepiopsis assectella (family Acrolepiidae),Agrilus plampennis (family Buprestidae), Anomala orientalis (familyScarabaeidae), Anoplophora chinensis, A. glabripennis (familyCerambycidae), Apis mellifera (family Apidae), Autographa gamma (familyNoctuidae), Bactrocera oleae (family Tephritidae), Bombus terrestris(family Apidae), Cactoblastis cactorum (family Pyralidae), Ceratitiscapitata (family Tephritidae), Cinara cupressi (family Aphididae),Coptotermes formosanus, C. gestroi (family Rhinotermitidae), Curculionucum, C. occidentis (family Curculionidae), Diuraphis noxia (familyAphididae), Dryocosmus kuriphilus (family Cynipidae), Epiphyaspostvittana (family Tortricidae), Eristalis tenax (family Syrphidae),Halyomorpha halys (family Pentatomidae), Helicoverpa armigera (familyNoctuidae), Homalodisca vitripennis (family Coccinellidae), Hylotrupesbajulus (family Cerambycidae), Incisitermes minor (familyKalotermitidae), Lasius neglectus or L. neoniger (family Formicidae),Leptinotarsa decemlineata (family Chrysomelidae), Leptoglossusoccidentalis (family Coreidae), Linepithema humile (family Formicidae),Lymantria dispar dispar (family Erebidae), Mamestra brassicae (familyNoctuidae), Monomorium destructor (family Formicidae), Monomoriumpharaonis (family Formicidae), Myrmica rubra (family Formicidae),Nylanderia fulva (family Formicidae), Operophtera brumata (familyGeometridae), Ostrinia nubilalis (family Crambidae), Oxycarenushyalinipennis (family Lygaeidae), Pectinophora gossypiella (familyGelechiidae), Pieris brassicae (family Pieridae), Polistes dominula(family Vespidae), Rhizotrogus majalis (family Scarabaeidae),Rhynchophorus ferrugineus (family Curculionidae), Schistocerca nitens(family Acrididae), Scirtothrips dorsalis (family Thripidae), Sirexnoctilio (family Siricidae), Sitophilus zeamais (family Curculionidae),Solenopsis invicta or S. saevissima (family Formicidae), Tetropspraeustus (family Cerambycidae), Thrips palmi (family Thripidae), Tremexfuscicornis (family Siricidae), Trogoderma granarium (familyDermestidae), Vespa velutina (family Vespidae), Vespula germanica(family Vespidae), Wasmannia auropunctata (family Formicidae), orXyleborus glabratus (family Curculionidae).

The natural pesticide may be applied in any appropriate manner. In someembodiments, a composition comprising the natural pesticides is appliedto a water body and its adjacent wetland areas infested with salvinias.In some embodiments, a composition comprising the natural pesticides isapplied to a structure (e.g., boats) to control salvinias. In someembodiments, a composition comprising the natural pesticides is appliedto a subject or structure (e.g., boats) to prevent infestation ofsalvinias. In some embodiments, a composition comprising the naturalpesticide is applied topically. In some embodiments, the naturalpesticide is applied to the surface of the invasive plant. In someembodiments, the composition is sprayed onto the invasive plant. In someembodiments, the composition is spread around the invasive plant. Insome embodiments, the composition is dissolved in water surrounding theinvasive plant.

In some embodiments, the application of the natural pesticide may notadd any exotic chemicals to the ecosystem of the invasive species. Insome embodiments, a composition comprising the natural pesticide can beprepared by chopping, shredding, blending, and/or pressing fresh matterof the target invasive species without solvent. In some embodiments, acomposition comprising the natural pesticide can be prepared by usingthe water in the treating water bodies and/or their adjacent wetlandareas as the solvent for the extraction of dried plant matter. In someembodiments, a composition comprising the natural pesticide can be addedwith non-bioactive surfactants.

The natural pesticide may be applied manually or mechanically. After thechopping, shredding, bending, and expression process, the residues fromthe juice production from fresh plant matter are usually not viable andare safe to be dumped back into the water body in a mechanicaloperation.

In some embodiments, the natural pesticide may be preferably applied tocontrol the invasive plant soon after it is produced. In someembodiments, the natural pesticide is best applied to control theinvasive plant immediately or within several hours of its production. Insome embodiments, the natural pesticide is preferably applied to controlthe invasive plant within a week of its production. In some embodiments,the natural pesticide especially in its aqueous form may be stored underrefrigeration (4° C.) for months and effectively inhibit the invasivespecies. In some embodiments, the natural pesticide especially in itssolid form may be stored under room temperature for months and caneffectively inhibit the invasive species.

In some embodiments, the natural pesticide may be applied alone. In someembodiments, the natural pesticide may be applied in combination withone or more secondary agents. In some embodiments, the secondary agentmay be formic acid, acetic acid, diquat (diquat dibromide), glyphosate,contact herbicides, other biocides, the Salvinia weevil, or biocontrolagents.

The natural pesticide may be present in any appropriate concentration inthe composition. In some embodiments, the composition contains 0.001,0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% byweight or more of the natural pesticide. In some embodiments, thecomposition contains about 0.01 to about 0.5% by weight of the naturalpesticide. In some embodiments, the composition contains about 0.1% byweight of endocide.

Also disclosed are the compositions as described herein, as well as kitscontaining the same.

When used in the context of a chemical group, “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “halo” means independently —F, —Cl,—Br or —I; “amino” means —NH₂; “hydroxyamino” means —NHOH; “nitro” means—NO₂; imino means ═NH; “cyano” means —CN; “isocyanate” means —N═C═O;“azido” means —N₃; in a monovalent context “phosphate” means —OP(O)(OH)₂or a deprotonated form thereof; in a divalent context “phosphate” means—OP(O)(OH)O— or a deprotonated form thereof; “mercapto” means —SH;“thio” means ═S; “sulfonyl” means —S(O)₂—; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond; and “≡” means triple bond. The symbol “----”represents an optional bond, which if present is either single ordouble. The symbol “

” represents a single bond or a double bond. Thus, for example, thestructure

includes the structures

As will be understood by a person of skill in the art, no one such ringatom forms part of more than one double bond. The symbol “

”, when drawn perpendicularly across a bond indicates a point ofattachment of the group. It is noted that the point of attachment istypically only identified in this manner for larger groups in order toassist the reader in rapidly and unambiguously identifying a point ofattachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the conformation (e.g., either R or S) orthe geometry is undefined (e.g., either E or Z).

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the groups and classes below, the following parenthetical subscriptsfurther define the group/class as follows: “(Cn)” defines the exactnumber (n) of carbon atoms in the group/class. “(C≤n)” defines themaximum number (n) of carbon atoms that can be in the group/class, withthe minimum number as small as possible for the group in question, e.g.,it is understood that the minimum number of carbon atoms in the group“alkenyl_((C≤8))” or the class “alkene_((C≤8))” is two. For example,“alkoxy_((C≤10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms). (Cn-n′) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group.Similarly, “alkyl_((C2-10))” designates those alkyl groups having from 2to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms)).

The term “saturated” as used herein means the compound or group somodified has no carbon-carbon double and no carbon-carbon triple bonds,except as noted below. The term does not preclude carbon-heteroatommultiple bonds, for example a carbon oxygen double bond or a carbonnitrogen double bond. Moreover, it does not preclude a carbon-carbondouble bond that may occur as part of keto-enol tautomerism orimine/enamine tautomerism.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound/group so modified is an acyclic or cyclic,but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by single bonds(alkanes/alkyl), or unsaturated, with one or more double bonds(alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl).When the term “aliphatic” is used without the “substituted” modifieronly carbon and hydrogen atoms are present. When the term is used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂,—OC(O)CH₃, or —S(O)₂NH₂.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched, cyclo, cyclic or acyclic structure,and no atoms other than carbon and hydrogen. Thus, as used hereincycloalkyl is a subset of alkyl. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (iso-Pr), —CH(CH₂)₂ (cyclopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(iso-butyl), —C(CH₃)₃ (tert-butyl), —CH₂C(CH₃)₃ (neo-pentyl),cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl arenon-limiting examples of alkyl groups. The term “alkanediyl” when usedwithout the “substituted” modifier refers to a divalent saturatedaliphatic group, with one or two saturated carbon atom(s) as thepoint(s) of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₂— (methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, —CH₂CH₂CH₂—, and

are non-limiting examples of alkanediyl groups. The term “alkylidene”when used without the “substituted” modifier refers to the divalentgroup ═CRR′ in which R and R′ are independently hydrogen, alkyl, or Rand R′ are taken together to represent an alkanediyl having at least twocarbon atoms. Non-limiting examples of alkylidene groups include: ═CH₂,═CH(CH₂CH₃), and ═C(CH₃)₂. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.The following groups are non-limiting examples of substituted alkylgroups: —CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH, —CH₂C(O)OCH₃,—CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂N(CH₃)₂,and —CH₂CH₂Cl. The term “haloalkyl” is a subset of substituted alkyl, inwhich one or more hydrogen atoms has been substituted with a halo groupand no other atoms aside from carbon, hydrogen and halogen are present.The group, —CH₂Cl is a non-limiting examples of a haloalkyl. An “alkane”refers to the compound H—R, wherein R is alkyl. The term “fluoroalkyl”is a subset of substituted alkyl, in which one or more hydrogen has beensubstituted with a fluoro group and no other atoms aside from carbon,hydrogen and fluorine are present. The groups, —CH₂F, —CF₃, and —CH₂CF₃are non-limiting examples of fluoroalkyl groups. An “alkane” refers tothe compound H—R, wherein R is alkyl.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one nonaromatic carbon-carbon double bond, nocarbon-carbon triple bonds, and no atoms other than carbon and hydrogen.Non-limiting examples of alkenyl groups include: —CH═CH₂ (vinyl),—CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl), —CH₂CH═CHCH₃, and—CH═CH—C₆H₅. The term “alkenediyl” when used without the “substituted”modifier refers to a divalent unsaturated aliphatic group, with twocarbon atoms as points of attachment, a linear or branched, cyclo,cyclic or acyclic structure, at least one nonaromatic carbon-carbondouble bond, no carbon-carbon triple bonds, and no atoms other thancarbon and hydrogen. The groups, —CH═CH—, —CH═C(CH₃)CH₂—, —CH═CHCH₂—,and

are non-limiting examples of alkenediyl groups. When these terms areused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂,—OC(O)CH₃, or —S(O)₂NH₂. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, arenon-limiting examples of substituted alkenyl groups. An “alkene” refersto the compound H—R, wherein R is alkenyl.

The term “alkynyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one carbon-carbon triple bond, and no atoms otherthan carbon and hydrogen. As used herein, the term alkynyl does notpreclude the presence of one or more non-aromatic carbon-carbon doublebonds. The groups, —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃, are non-limitingexamples of alkynyl groups. When alkynyl is used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. An“alkyne” refers to the compound H—R, wherein R is alkynyl.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl group (carbon number limitation permitting) attached tothe first aromatic ring or any additional aromatic ring present.Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl,(dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, and themonovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic group,with two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl group (carbon number limitation permitting) attached to the firstaromatic ring or any additional aromatic ring present. If more than onering is present, the rings may be fused or unfused. Non-limitingexamples of arenediyl groups include:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. An “arene” refers to thecompound H—R, wherein R is aryl.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples of aralkyls are: phenylmethyl(benzyl, Bn) and 2-phenyl-ethyl. When the term is used with the“substituted” modifier one or more hydrogen atom from the alkanediyland/or the aryl has been independently replaced by —OH, —F, —Cl, —Br,—I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. Non-limiting examples ofsubstituted aralkyls are: (3-chlorophenyl)-methyl, and2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein,the term does not preclude the presence of one or more alkyl, aryl,and/or aralkyl groups (carbon number limitation permitting) attached tothe aromatic ring or aromatic ring system. If more than one ring ispresent, the rings may be fused or unfused. Non-limiting examples ofheteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im),isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl,pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl,triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term“heteroarenediyl” when used without the “substituted” modifier refers toan divalent aromatic group, with two aromatic carbon atoms, two aromaticnitrogen atoms, or one aromatic carbon atom and one aromatic nitrogenatom as the two points of attachment, said atoms forming part of one ormore aromatic ring structure(s) wherein at least one of the ring atomsis nitrogen, oxygen or sulfur, and wherein the divalent group consistsof no atoms other than carbon, hydrogen, aromatic nitrogen, aromaticoxygen and aromatic sulfur. As used herein, the term does not precludethe presence of one or more alkyl, aryl, and/or aralkyl groups (carbonnumber limitation permitting) attached to the aromatic ring or aromaticring system. If more than one ring is present, the rings may be fused orunfused. Non-limiting examples of heteroarenediyl groups include:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The term “heterocycloalkyl” when used without the “substituted” modifierrefers to a monovalent non-aromatic group with a carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of one or more non-aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heterocycloalkyl group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. As used herein, the term does notpreclude the presence of one or more alkyl groups (carbon numberlimitation permitting) attached to the ring or ring system. If more thanone ring is present, the rings may be fused or unfused. Non-limitingexamples of heterocycloalkyl groups include aziridinyl, azetidinyl,pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, andpyranyl. When the term “heterocycloalkyl” used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, aryl, aralkyl orheteroaryl, as those terms are defined above. The groups, —CHO, —C(O)CH₃(acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃, —C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂,—C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅, —C(O)(imidazolyl) arenon-limiting examples of acyl groups. A “thioacyl” is defined in ananalogous manner, except that the oxygen atom of the group —C(O)R hasbeen replaced with a sulfur atom, —C(S)R. When either of these terms areused with the “substituted” modifier one or more hydrogen atom(including the hydrogen atom directly attached the carbonyl orthiocarbonyl group) has been independently replaced by —OH, —F, —Cl,—Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃,—C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. The groups,—C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃ (methylcarboxyl), —CO₂CH₂CH₃,—C(O)NH₂ (carbamoyl), and —CON(CH₃)₂, are non-limiting examples ofsubstituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃ (methoxy),—OCH₂CH₃ (ethoxy), —OCH₂CH₂CH₃, —OCH(CH₃)₂ (isopropoxy), —OCH(CH₂)₂,—O-cyclopentyl, and —O-cyclohexyl. The terms “alkenyloxy”, “alkynyloxy”,“aryloxy”, “aralkoxy”, “heteroaryloxy”, and “acyloxy”, when used withoutthe “substituted” modifier, refers to groups, defined as —OR, in which Ris alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and acyl, respectively.The term “alkoxydiyl” refers to the divalent group —O— alkanediyl-, —O—alkanediyl-O—, or -alkanediyl-O-alkanediyl-. The term “alkylthio” and“acylthio” when used without the “substituted” modifier refers to thegroup —SR, in which R is an alkyl and acyl, respectively. When any ofthese terms is used with the “substituted” modifier one or more hydrogenatom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂,—NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂,—C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. The term “alcohol” corresponds to analkane, as defined above, wherein at least one of the hydrogen atoms hasbeen replaced with a hydroxy group.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃ and —NHCH₂CH₃. The term “dialkylamino” when used without the“substituted” modifier refers to the group —NRR′, in which R and R′ canbe the same or different alkyl groups, or R and R′ can be taken togetherto represent an alkanediyl. Non-limiting examples of dialkylamino groupsinclude: —N(CH₃)₂, —N(CH₃)(CH₂CH₃), and N-pyrrolidinyl. The terms“alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, and “alkylsulfonylamino” when usedwithout the “substituted” modifier, refers to groups, defined as —NHR,in which R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, andalkylsulfonyl, respectively. A non-limiting example of an arylaminogroup is —NHC₆H₅. The term “amido” (acylamino), when used without the“substituted” modifier, refers to the group —NHR, in which R is acyl, asthat term is defined above. A non-limiting example of an amido group is—NHC(O)CH₃. The term “alkylimino” when used without the “substituted”modifier refers to the divalent group ═NR, in which R is an alkyl, asthat term is defined above. The term “alkylaminodiyl” refers to thedivalent group —NH-alkanediyl-, —NH-alkanediyl-NH—, or-alkanediyl-NH-alkanediyl-. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ are non-limiting examples ofsubstituted amido groups.

The terms “alkylsulfonyl” and “alkylsulfinyl” when used without the“substituted” modifier refers to the groups —S(O)₂R and —S(O)R,respectively, in which R is an alkyl, as that term is defined above. Theterms “alkenylsulfonyl”, “alkynylsulfonyl”, “aryl sulfonyl”, “aralkylsulfonyl”, and “heteroarylsulfonyl”, are defined in an analogous manner.When any of these terms is used with the “substituted” modifier one ormore hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br,—I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

As used herein, a “chiral auxiliary” refers to a removable chiral groupthat is capable of influencing the stereoselectivity of a reaction.Persons of skill in the art are familiar with such compounds, and manyare commercially available.

The term “invasive species” means a species (e.g., plants and animalsincluding insects) that is either native or non-native (exotic) to theecosystem and whose presence or introduction causes or likely causeseconomical or environmental harm or harm to human health.

“Invasive plant species” or “invasive plant” means either a non-native(exotic) or native invasive plant species.

“Invasive aquatic species” or “aquatic invasive species” means aninvasive species that has living in, on, or next to water.

“Invasive aquatic plant” means an invasive plant species that hasadapted to living in, on, or next to water, and that can grow eithersubmerged or partially submerged in water.

“Water body” or “body of water” means any significant accumulation ofwater on a planet's surface, including but not limited to lake, pond,river, canal, creek, stream, brook, channel, ditch, bay, bayou, swamp,marsh, slough, bog, fen, wetland, harbor, inlet, lagoon, puddle,reservoir, strait, spring, swimming pool, or any container or structurewith permanent or seasonal water.

“Salvinias” means aquatic or semi-aquatic fern species of the orderSalviniales, including families Salviniaceae Reichenbach (SalviniaSéguier), Azollaceae Wettstein (Azolla Lamarck) (sometimes, Azolla istreated as a genus of the family Salviniaceae), and Marsileaceae Mirbel(Marsilea L., Pilularia L., and Regnellidium Lindm).

“Primary stage” or “primary growth stage” of giant Salvinia means theplant growth occurs in the early stages of an infestation. The small,flat, and oval-shaped floating leaves lie in direct are less than 15 mmin width.

“Secondary stage” or “secondary growth stage” of giant Salvinia meansthe plant growth occurs in the secondary stages of an infestation. Thefloating leaves become slightly cupped and are more than 15 mm but lessthan 50 mm in width.

“Tertiary stage” or “tertiary growth stage” of giant Salvinia means theplant growth occurs in the mature stages of an infestation. The floatingleaves become tightly folded and are more than 50 mm in width whenforced open. Tertiary plants may form a multilayered mat on the surfaceof the infested water body.

“Gland” means a cell, group of cells, or organ producing a secretion.“Exocrine gland” means any gland that secretes its products through aduct onto an epithelial surface.

“Trichome” refers to “glandular trichome” or plant gland in thisinvention and means glandular unicellular or multicellular appendages onthe surface of various plant organs.

“Effective” amount or concentration means that amount or concentrationwhich, when applied to a place or subject for controlling invasivespecies, is sufficient to affect the growth, reproduction, or spread ofthe species.

“Control” or “controlling” means inhibiting growth, reproduction, orspread of an invasive species in a place or subject. As used herein, theterm “inhibition” of the species also refers to slowing, interrupting,arresting or stopping the species and does not necessarily indicate atotal elimination of the species.

Some abbreviations used herein are as follows: ¹H-NMR is proton nuclearmagnetic resonance, ¹³C-NMR is carbon nuclear magnetic resonance,ADEQUATE is adequate sensitivity double-quantum spectroscopy, Al isaluminum, Ag is silver, B is boron, Ba is barium, Ca is calcium, As isarsenic, Cd is cadmium, HCOOH is formic acid, CHCl₃ is chroroform,CH₂C₁₂ is dichloromethane or methylene chloride, CH₃COOH is acetic acid,CH₃CN is acetonitrile, Co is cobalt, COSY is correlation spectroscopy,Cr is chromium, Cu is copper, DMSO is dimethyl sulfoxide, EC₅₀ meanshalf maximal effective concentration, EtOH is ethanol, FAB MS is fastatom bombardment mass spectroscopy, Fe is iron, g is gram(s), gal isgallon(s), Glc is glucose, h is hour(s), H₂O is water, Hg is mercury,HMBC is heteronuclear multiple-bond correlation, HPLC is highperformance liquid chromatography, HRESIMS is High-resolutionelectrospray ionization mass spectrometry, HSQC is heteronuclear singlequantum coherence spectroscopy, K is potassium, kg is kilogram(s), kPais kilopascal(s), ICP is inductively coupled plasma, L is liter(s), LD₅₀is defined as the dose required to kill half of the exposed fire ants,LD₉₀ is defined as the dose required to kill 90% of the exposed fireants, m is meter(s), MeOH is methanol, mg is milligram, min isminute(s), Mg is magnesium, mL is milliliter(s), mm is millimeter(s), Mnis manganese, Mo is molybdenum, Na is sodium, NCPC is National Centerfor Pharmaceutical Crops of the Arthur Temple College of Forestry andAgriculture at Stephen F. Austin State University in Nacogdoches, Tex.,USA, NMR is nuclear magnetic resonance, P is phosphorus, Pb is lead, pHis a measure of the acidity or basicity of an aqueous solution, psi ispounds per square inch, ppt is parts per thousand, S is sulfur, Se isselenium, sec is second(s), RT is room temperature (approximately at 20°C.), “tea bag” is a bag of ground dried plant matter, “tea brick” iscompressed brick or pie of ground dried plan matter without bag, Zn iszinc, μg is microgram(s), μL is microliter(s), and μM is micromolar(s).

The terms “about” or “approximately” are defined as being close to asunderstood by one of ordinary skill in the art, and in one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

The use of the word “a” or “an” when used in conjunction with the term“comprising” may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

The words “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not excludeadditional, unrecited elements or method steps.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification. Compositions and methods“consisting essentially of” any of the ingredients or steps disclosedlimits the scope of the claim to the specified materials or steps whichdo not materially affect the basic and novel characteristic of theclaimed invention.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—The scheme shows the discovery and development process andrelated experiments of endocides from invasive giant Salvinia (Salviniamolesta) as described herein (inactive: not able to inhibit the growthof giant Salvinia at all; active: able to fully inhibit the growth ofgiant Salvinia at high concentrations; very active: able to fullyinhibit the growth of giant Salvinia at low concentrations).

FIG. 2—Salvinia species are rootless, free-floating aquatic ferns andthey are well-known for their extremely water-repellent floating leaves.On the upper surfaces of the floating leaves of S. molesta, fourmulticellular glandular trichomes (called “trichomes”) have their apicalcells connected to form egg-beater structures. The three short arrowspoint to the egg-beater structures of trichomes which were removed usinga blade. The root-like submerged leaves are highly branched withmulticellular trichomes on which sporocarps develop.

FIG. 3—Effects of dry plant matter of giant Salvinia (Salvinia molesta)(“tea bag”) on the growth of giant Salvinia in containers during thefour weeks of greenhouse experiments (with bars presenting standarddeviations).

FIG. 4—The photograph shows that giant Salvinia (Salvinia molesta)plants were totally eliminated by the dried plant matter of giantSalvinia (a bag of 400 g plant matter was placed in the bottom of eachof the containers on the bottom row) by the end of the second week ofexperiments in the greenhouse in comparison with controls (containers onthe top row); there was no new growth within the six months ofobservation.

FIG. 5—The photograph shows that the root-like submerged leaves of giantSalvinia (Salvinia molesta) were totally damaged before the floatingleaves turning into brown and dead after the “tea bag” treatment.

FIG. 6—The photograph shows the wilted trichomes on the upper surfacesof the floating leaves of giant Salvinia (Salvinia molesta) caused bythe water extract from “tea bag” (left) in comparison with those in thenormal plant growth condition (right). The wilted trichomes releasedautotoxic compounds and enhanced the endocidal function of “tea bag”.

FIG. 7—The photograph shows that all giant Salvinia (Salvinia molesta)plants were eliminated within the 13 weeks after the treatment withwater extract of the dried matter of giant Salvinia. The plants in thetop row served as control without any treatment, and each plant hasdeveloped significant new growth. The plants in the middle row weretreated with 0.01% water extract of giant Salvinia, and some plants havedeveloped new growth. The plants in the bottom row were treated with0.1% water extract of giant Salvinia, and all plants are dead and no newgrowth was developed from any plants during the eight months ofobservation. The three columns represent three replications of theexperiment.

FIG. 8—The photograph showing growth performance of giant Salvinia(Salvinia molesta) plants two weeks after the treatments of waterextract of the dried matter of giant Salvinia and its three fractions incomparison with those in the control group. Top row: plants cultured inwater without any treatment (control); 2nd row: plants were treated with0.1% MeOH fraction (all plants dead); 3rd row: plants were treated with0.1% MeOH/CH₂Cl₂ (1:1) fraction; 4th row: plants were treated with 0.1%H₂O fraction; and the bottom row: 0.1% water extract of giant Salvinia.The three columns represent three replications of the experiment.

FIG. 9—Structure of bioactive salviniside II as endocide isolated fromgiant Salvinia (Salvinia molesta).

FIG. 10—¹H and ¹³C-NMR Data (δ) of salviniside II.

FIG. 11—Giant Salvinia (Salvinia molesta) can be totally eliminated bysalviniside II isolated from giant Salvinia within two weeks (with barspresenting standard deviations).

FIG. 12—HPLC profiles of water extract from the dried matter of giantSalvinia (Salvinia molesta) on the preparation day (upper) and storageas the powder at RT for 26 weeks (bottom) using Agilent 1100 HPLC system(Eclipse XDB-C18 column (4.6×150 mm, 3.5 μM) at a flow rate of 0.6mL/min, detection wavelength=254 nm, the extract concentration: 24 kg/Lin fresh weight, and the injection volume: 3 μL). The stored powder hadapproximately 70% of the total peak area of that in the newly preparedextract.

FIG. 13—HPLC profiles of 5% H₂O fraction of water extract from the driedmatter of giant Salvinia (Salvinia molesta) on the preparation day(upper) and stored in refrigerator (4° C.) for 26 weeks (bottom) usingAgilent 1100 HPLC system (Eclipse XDB-C18 column (4.6×150 mm, 3.5 μM) ata flow rate of 0.6 mL/min, detection wavelength=254 nm, the extractconcentration: 16 kg/L in fresh weight, and the injection volume: 50μL). The stored H₂O fraction still had approximately 90% of the chemicalcontents of the newly prepared sample.

FIG. 14—The growth of giant Salvinia (Salvinia molesta) can besuccessfully controlled by the endocide treatments with or withoutsurfactants. By the end of the third week, all plants treated witheither “tea bag” of the dried matter, water extract of the dried matterwith surfactants, or H₂O fraction of the water extract from the driedmatter with surfactants were dead and there was no new growth during thefollowing four months of observation. By the end of the 4^(th) week,over 90% or 95% of the plants treated with water extract and H₂Ofraction of the water extract were dead, respectively (with barspresenting standard deviations).

FIG. 15—The photograph shows that all giant Salvinia (Salvinia molesta)plants were eliminated within three weeks after the treatment using a“tea bag” of the dried matter of giant Salvinia (right) in comparisonwith control (left).

FIG. 16—The growth of giant Salvinia (Salvinia molesta) can besuccessfully controlled by the extract juice from the fresh matter ofgiant Salvinia by hydraulic press. After two treatments, the hydraulicextract juice with surfactants totally eliminated the giant Salviniawithin four weeks and there was no new growth thereafter (with barspresenting standard deviations).

FIG. 17—The photograph shows that all giant Salvinia (Salvinia molesta)plants were eliminated within four weeks after the treatment withhydraulic extract juice from the fresh matter of giant Salvinia (right)in comparison with the control (left).

FIG. 18—The photograph shows that the trichomes on the upper surfaces ofthe floating leaves of giant Salvinia (Salvinia molesta) weresignificantly damaged by the hydraulic extract (right) in comparisonwith those in the control (left). The wilted trichomes caused by thehydraulic extract released autotoxic chemicals and enhanced theendocidal function of the hydraulic extract.

FIG. 19—The photograph shows that there was no plant survival or newgrowth in the cultured residues from the juice production from freshplant matter of giant Salvinia (Salvinia molesta) within six weeks(white dots are living least duckweed (Lemna minuta)).

FIG. 20—The diagram shows that both water extract from the dried matterand hydraulic extract from the fresh matter of giant Salvinia withsurfactants effectively inhibited the giant Salvinia after twotreatments (with bars presenting standard deviations).

FIG. 21—HPLC profiles of water extract from the dried matter of giantSalvinia (Salvinia molesta) (upper) and hydraulic extract from freshmatter of giant Salvinia (bottom) by using an Agilent 1100 HPLC systemcoupled to an Agilent 1100 diode array detector, and an Eclipse XDB-C18column (4.6×150 mm, 3.5 μM) at a flow rate of 0.6 mL/min. A gradientelution was performed by using H₂O (A) and CH₃CN (B) as mobile phases.Elution was performed according to the following conditions: 2% B attime 0, linear increase to 98% B in 22 min, and hold 98% B for 8 min.The column temperature was maintained at 23° C. The HPLC chromatogramwas standardized on retention times and peak intensities of the peaksobserved at a wavelength of 254 nm. The concentration of both waterextracts and hydraulic extract were 2 kg fresh plant matter/L. Theinjection volume was 50 μL for all analyses.

FIG. 22—HPLC profiles of water extract from the dried matter of giantSalvinia (Salvinia molesta) (upper) and hydraulic extract from the freshmatter of giant Salvinia (bottom) by using Agilent 1260 HPLC systemcoupled to an Agilent 1260 diode array detector, and an Acclaim HILIC-10column (4.6×150 mm, 3.0 μM) at a flow rate of 0.6 mL/min. The mobilephase was CH₃CN/H₂O (65:35, v/v). The column temperature was maintainedat 23° C. The HPLC chromatogram was standardized on retention times andpeak intensities of the peaks observed at a wavelength of 230 nm. Theconcentration of both water extracts and hydraulic extract were 40 kgfresh plant matter/L. The injection volume was 20 for all analyses.

FIG. 23—The diagram shows that the hydraulic extracts are more stablewhen stored under refrigeration (4° C.) than that at RT. The total peakarea of hydraulic extract stored at refrigeration (4° C.) for one, two,and 14 weeks contained 70%, 66%, and 60% of the total chemical contentsin the newly prepared extract, respectively (with bars presentingstandard deviations).

FIG. 24—HPLC profiles of hydraulic extract from the fresh matter ofgiant Salvinia (Salvinia molesta) on the preparation day (upper) andstored at refrigerator (4° C.) for 14 weeks (bottom) by using Agilent1100 HPLC system (Eclipse XDB-C18 column (4.6×150 mm, 3.5 μM) at a flowrate of 0.6 mL/min, detection wavelength=254 nm, the extractconcentration: 2 kg/L in fresh weight, and the injection volume: 50 μL).

FIG. 25—Metal analysis of the dried plant matter, water extract powder,and its precipitated salts of giant Salvinia (Salvinia molesta) byThermo Scientific ICAP 7200 ICP-OES.

FIG. 26—The diagram shows that the growth of the floating fern (Salviniaminima) plants can be totally controlled by the water extract of driedmatter of giant Salvinia (Salvinia molesta) within six weeks (with barspresenting standard deviations).

FIG. 27—The diagram shows that the plants of Carolina mosquito fern(Azolla caroliana) was inhibited by the water extract at higherconcentration (2.5% or higher, in dry weight) (with bars presentingstandard deviations).

FIG. 28—The photograph shows that all giant Salvinia (Salvinia molesta)plants were eliminated within 13 weeks after the treatment with waterextract of the dried matter of least duckweed (Lemna minuta). The plantsin the top row served as control without any treatment, and each planthas developed significant new growth. The plants in the middle row weretreated with 0.01% water extract of duckweed, and some plants havedeveloped new growth. The plants in the bottom row were treated with0.1% water extract of duckweed, and all plants are dead and no any newgrowth was developed from any plants during the eight months ofobservation. The three columns represent three replications of theexperiment.

FIG. 29—The diagram shows the new growth per plant of giant Salvinia(Salvinia molesta) by the end of the 13th week after treatment withwater or ethanol extract of dried matter of least duckweed (Lemnaminuta) in comparison with untreated plants (control) (with barspresenting standard deviations).

FIG. 30—The picture shows that all giant Salvinia (Salvinia molesta)plants were eliminated within 13 weeks after treatment with ethanolextract of the dried matter of least duckweed (Lemna minuta). The plantsin the top row served as control without any extract treatment, and eachplant has developed significant new growth. The plants in the middle rowwere treated with 0.01% ethanol extract of duckweed, and plants havedeveloped new growth. The plants in the bottom row were treated with0.1% ethanol extract of duckweed, and 12 plants are dead and only 3plants have developed any new growth. However, all these 15 plantstreated with 0.1% ethanol extract of least duckweed were dead by the endof 15 weeks of the experiment and no new growth occurred thereafterduring the eight months of observation. The three columns representthree replications of the experiment.

FIG. 31—The photograph shows that some floating fern (Salvinia minima)plants were eliminated within the 13 weeks after the treatment withwater extract of the dried matter of least duckweed (Lemna minuta). Theplants in the top row served as control without any extract treatment.The plants in the middle row were treated with 0.01% ethanol extract ofduckweed. The plants in the bottom row were treated with 0.1% ethanolextract of duckweed. The three columns represent three replications ofthe experiment.

FIG. 32—The diagram shows new growth per plant of floating fern(Salvinia minima) by the end of the 13th week after treatment with wateror ethanol extract of the dried matter of least duckweed (Lemna minuta)in comparison with untreated plants (control) (with bars presentingstandard deviations).

FIG. 33—The photograph shows that all floating fern (Salvinia minima)plants were eliminated by the end of 13 weeks after the treatment with0.1% ethanol extract of the dried matter of duckweed (Lemna minuta). Theplants in the top row served as control without any treatment, and thereis significant new plant growth. The plants in the middle row weretreated with 0.01% ethanol extract of duckweed, and new growth can beobserved. The plants in the bottom row were treated with 0.1% ethanolextract of duckweed, and some plants are dead and minimal new growth canbe observed. The three columns represent three replications of theexperiment.

FIG. 34—The photograph shows that 3-week-old Brazilian pepper tree(Schinus terebinthifolius Raddi) seedlings had significant damage afterthe first treatment with 10% Brazilian pepper tree extract or Brazilianpepper tree extract with surfactants in comparison with those in controland surfactants treatment. By the end of the 4th week, all six peppertree seedlings were dead following the pepper tree extract withsurfactants treatment and five seedlings were dead and one had deadapical bud and young leaves after treated by the pepper tree extract.

FIG. 35—The photographs show that 3-week-old seedlings of Chinese tallowtree (Triadica sebifera (L.) Small) and Chinese privet (Ligustrumsinense Lour.) (family Oleaceae) under treatment with 10% Chinese tallowtree fruit extract. All six Chinese tallow tree seedlings treated by theethanol extract of Chinese tallow tree fruits were significantly damaged(right on the upper row) in comparison with control (left on the upperrow). However, the tollowtree extract did not cause any damage inChinese privet seedlings (right on the bottom row) while the Chineseprivet extract did not damage the tallow tree seedlings (left on thebottom row).

FIG. 36—The diagram shows the impacts of acetone extract and ethanolextract of the red imported fire ant (Solenopsis invicta Buren) on theworkers of the red imported fire ant. The ethanol extract showed moresignificant toxicity against the fire ants than the acetone extract.During the 7 h of contact toxicity bioassays, an average ofapproximately 60%, 70%, or 80% of the 50 fire ants in contact with 1%,5%, or 10% ethanol extract were dead, respectively.

FIG. 37—The diagram shows the impacts of ethanol extract of the redimported fire ants and formic acid on the workers of the red importedfire ant (Solenopsis invicta Buren). During the 90 min of contacttoxicity bioassays, the combined applications of ethanol extract andformic acid had more significant effects than the use of either ethanolextract or formic acid alone (with bars presenting standard deviations).

FIG. 38—The diagram shows the impacts of ethanol extract of the redimported fire ant (Solenopsis invicta Buren) and formic acid on theinjured workers of the red imported fire ants. During the 90 min ofcontact toxicity bioassays, the combined applications of ethanol extractand formic acid had more significant effects than the use of eitherethanol extract or formic acid alone (with bars presenting standarddeviations).

FIG. 39—The diagram shows the occurrence of formic acid (HCOOH) inchroroform (CHCl₃) extract of the subterranean termite (Reticulitermesflavipes (Kollar)) by NMR analysis. ¹H-NMR experiments were performed ona JEOL ECS 400 spectrometer, with spectroscopic data referenced to thesolvent used. According to standard formic acid ¹H-NMR spectrum, theunique singlet of the aldehyde proton should appear at δ_(H) 8.02.

FIG. 40—The diagram shows the impacts of formic acid on the subterraneantermite (Reticulitermes flavipes (Kollar)). Formic acid at higherconcentrations (5 or 10%) killed all termites (including workers,soldiers, and reproductive) during the first hour of contact experiment.More than 40% of the termites were killed by the 1% formic acid duringthe 7 h of contact toxicity bioassays. However, formic acid at 0.1%concentration had no impact on the termites (with bars presentingstandard deviations).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Control of invasive species by its endogenous metabolites has never beenproposed or used.

The inventors recently discovered that some aquatic plants, trees, fireants, or termites experienced slow autocide by their own toxins thatwere released into the environment by themselves or other individualswithin the same species. It was known that some bioactive secondarymetabolites may accumulate in glands (e.g., epidermal glandulartrichomes of plants or exocrine glands of insects). These compoundsusually do not interrupt normal growth of the producing species due totheir non-bioavailability. However, it was discovered that externalapplication of these bioactive agents to the parent species will inhibitand even eliminate the species. Further, the parent species may be moresensitive to its endogenous toxic metabolites than other species.

As a proof of principle, the inventors found that endocides from giantSalvinia and extracts of least duckweed (Lemna minuta) (Lernnaceae) caneliminate existing giant Salvinia quickly and prohibit plant growth forat least six months in controlled conditions. The experiments showedthat the effective materials used to extract bioactive agents to controlSalvinia spp. include any ground dry biomass matters of Salvinia andLemna. Experiments also found that the fresh biomass of Salvinia andLemna, particularly chopped can also be useful to extract bioactiveagents or direct application in control of Salvinia spp. Raw andunground dry plant matter can also be used in a similar manner, and itis believed that any other plant species containing the active agents asdescribed herein could be useful in production of biocides to controlSalvinia spp. This method is useful for controlling invasive aquaticplants, but this method also has potential in territorial plants andendophytic fungi. It is especially useful for controlling invasiveplants with trichomes as the primary accumulation sites of bioactivecompounds.

Salvinia species are rootless, free-floating aquatic ferns and they arewell-known for their extremely water-repellent floating leaves. On theupper surfaces of the floating leaves of S. molesta, four multicellularglandular trichomes have their apical cells connected to form egg-beaterstructures. The arrows point to the egg-beater structures of trichomeswhich were removed by blade. The root-like submerged leaves are highlybranched with multicellular trichomes and on which sporocarps developed.

Previous experiments found that the active agents of giant Salvinia areprimarily accumulated in trichomes which are usually intact, and theplant can avoid poison by these toxins (Li et al., unpublished data).However, Salvinia spp. are well-known for their water-repellent floatingleaves because of the leaves are densely covered with peculiar trichomes(Barthlott et al. 2009). On the upper surfaces of floating leaves of S.molesta, four multicellular trichomes have their apical cells connectedto form egg-beater structures (FIG. 2). The root-like submergered leavesalso have dense multicellular trichomes (FIG. 2). The basal cells of thetrichomes in S. molesta are covered with wax (Barthlott et al. 2009).Therefore, the bioactive compounds in trichomes of the living plants arenot released into the water because of this water-repellent feature. Theremoval or damage of the apical cells (egg-beater structures) oftrichomes (FIG. 2) will not only release the chemicals in storage butalso allow water to access the floating leaves to soak the bioactiveagents out. This invention emphasizes that breaking the trichomes (e.g.,mechanical treatments such as grinding, blending, squeezing, or heavilywashing plant tissues) is necessary for effective extraction of thebioactive agents from giant Salvinia.

Both water and organic solvents (e.g., ethanol) can be used toeffectively extract the bioactive agents. The most effective solvent todissolve these bioactive entities, fractions, or extracts to controlSalvinia spp. is water, but organic solvents could also be useful inapplication. Plants respond to the treatment more quickly at highertemperatures than at lower temperatures (RT).

The endocidal functions are not only in giant Salvinia, a fern speciesin the phylum Pteridophyta of the kingdom Plantae, but also discoveredin Brazilian pepper tree and Chinese tallow tree, two tree species inthe phylum Flowering Plants of the kingdom Plantae and the red importedfire ant and subterranean termite, two insect species from the phylumArthropoda of the kingdom Animalia. The plant growth of Brazilian peppertree seedlings had significant damage by the 10% ethanol extract ofBrazilian pepper tree fruits after the first treatment (FIG. 34). Fourweeks later, all six Brazilian pepper tree seedlings were killed by theextract with surfactants and five of the six seedlings treated by theextract alone were dead with one significantly injured. The seedlings ofsweetgum (Liquidambar styraciflua L.) (family Altingiaceae) and Shumardoak (Quercus shumardii Buckland) (family Fagaceae) as well as poison ivy(Toxicodendron radicans (L.) Kuntze or Rhus toxicodendron L.) of thesame family with Brazilian pepper tree were not impacted by theBrazilian pepper tree extract. Furthermore, the Brazilian pepper treeseedlings were no inhibited by either surfactants or the 10% ethanolextract of Chinese tallow tree (Triadica sebifera (L.) Small) fruits.

The ethanol extract of the fruits of Chinese tallow tree (Triadicasebifera (L.) Small) (Eurporbiaceae) inhibited and even killed youngseedlings of the tallow tree. However, the tollowtree extract did notcause any damage in the seedlings of Chinese privet (Ligustrum sinenseLour.) of the family Oleaceae. Further, the Chinese privet extract didnot damage the tallow tree seedlings.

Formic acid commonly occurs in ants (family Formicidae), termites(Isoptera), and some other insects. The organic acid is accumulated inexocrine glands of these insects and serves as defensive weapon fightingagainst attackers. The formic acid can be as concentrated as 60% of thesecretion of ants, and workers can contain as much as 2 mg each (Morgan2008). The acid is a known natural pesticide. Like their enemies,however, both the red imported fire ants and subterranean termites couldnot avoid toxicity of formic acid during external topical or fumigationapplication (FIGS. 37-40).

This invention found that the red imported fire ants can be killed bythe extracts of the fire ants (without formic acid), particularlyethanol extract (FIG. 36). However, the ethanol extract (primarilypiperidine alkaloids) of the fire ants had selective activity againstthe ants and showed no effects on the subterranean termite. The fireants and termites are in different superorders (Endopterygota andExopterygota, respectively).

Both formic acid and acetic acid have been used as herbicides to controlweeds. This invention found that formic acid is more effective thanacetic acid to control giant Salvinia. Formic acid at 0.05%concentration killed approximately 50% of the giant Salvinia plantswithin 24 h. It is more effective in Salvinia control when combined withgiant Salvinia extracts.

This invention reports that bioactive compounds in giant Salvinia areprimarily accumulated in glandular trichomes on the leaf surface. It isknown that the surface of the fruits (drupes) of Brazilian pepper treeis also covered by glandular trichomes (Carmello-Guerreiro and Paoli2002). Chinese tallow tree has glands on leaf stalks. Formic acid is aprimary venom compound commonly accumulated in the abdominal exocrineglands of ants and termites. In all these five described distant-relatedspecies, glands act accumulation sites of bioactive compounds that playimportant roles in endocidal function of the species so that thebioactive compounds do not poison the producing species in normalgrowth. Thus, the endocide may have effects especially in the specieswith glands as accumulation sites of bioactive compounds.

Furthermore, this invention disclosed that application of endocides atlow concentrations (dosages) can control partial growth of an individual(e.g., a specific cell(s), tissue(s), or organ(s)) of the producingspecies rather than killing or eliminating the species.

Application of endocides at low concentrations (dosages) may produceabnormal morphogenesis. In some embodiments, the endocides may inducemutations. In some embodiments, the induced mutations may be used toproduce desirable genotypes.

In some weeds and crops, autotoxicity reports are primarily on effectsin the seed germination, growth, and yield of the producing plants andthe resulting soil sickness and replanting problems. The reportedautotoxic chemicals had weak activity against the parent species butthey are more potent inhibitory effects on other non-closely-relatedspecies (e.g., species in other genera, families or orders) than on theproducing plants. Therefore, it is impossible to develop a biocidalproduct to selectively control the producing invasive species. Further,it is commonly believed that a species can avoid self-toxicity by itsendogenous toxic metabolites and the toxic compounds are primarily usedfor defense to protect itself from enemies. The present inventiondiscovered that some invasive species with glands as accumulation siteof their autotoxic chemicals cannot avoid autotoxicity by its ownmetabolite(s) when externally applied. More importantly, these parentspecies (and its closely-related species) are more sensitive to itsendogenous toxic metabolites than the other tested species in differentgenera, families or orders. The mechanism of selective activity of theautotoxic chemicals to the producing species is not clear but it seemsthat these chemicals in glands are not available to the normal growthand development of the species.

This discovery provides the foundation for development of a novelendocide product to selectively control an invasive species over theother species. It further suggests that how to avoid endogenousautotoxicity by the toxic metabolite(s) must be the priority for theproducing species in the development and evolution. To protect itselffrom enemies by “defensive” metabolite(s) become possible only when theproducing species can avoid endogenous autotoxicity.

A. Natural Pesticide Compositions

1. Endocides

In some embodiments, the present invention relates to compositionscontaining endocides and methods of controlling the growth of invasivespecies using the same. An endocide (endogenous biocide) is a biocidederived from an endogenous bioactive agent (e.g., secondary metabolite)that does not cause apparent poison in normal growth of the producingspecies but will poison or inhibit and even eliminate the parent specieswhen induced in producing species. It can selectively eliminate theparent species (and possibly its closely-related species) whenexternally applied. The dead tissues of species caused by an endocidewill enhance the endocidal function to the species. The endocide can bedeveloped as either a pure single entity or as a mixture of compounds(e.g., a fraction of extract, an extract, a dry or fresh matter ofspecies). The endocides can be obtained in liquid or solid format fromfresh or dried matter by any effective extraction methods (e.g.,expression, distillation, solvent extraction, infusion, decoction, andpercolation). For aquatic species, endocides are usually water solubleand can be dissolved in water in application. Endocides may have effectson the invasive species in all or some growth stages and all or sometissues.

The endocide development process is summarized in FIG. 1. As an example,the inventors studied giant Salvinia plants. It was found that endocidesderived from either dried or fresh plant matter of giant Salvinia caneffectively control the invasive species in all growth stages (primary,secondary, and tertiary stages) (Examples 1-8). It was found that dryplant matter of giant Salvinia can fully eliminate giant Salvinia plantswhen adequate dried ground matter of giant Salvinia is placed in thewater (FIG. 3).

FIG. 3 illustrates that the minimum effective concentration to inhibitgiant Salvinia is between 4 and 8 g of dried plant matter per liter ofwater. About 50% of giant Salvinia plants were killed at theconcentration of 8 g/L. The Salvinia plants were fully eliminated at 16g/L within two weeks in the greenhouse (FIGS. 3 and 4), and no new orliving Salvinia plants were found within the six months of observationthereafter. This demonstrates that giant Salvinia contains some agentsthat are toxic to the parent plants and that these agents can fullyeliminate and inhibit the growth of Salvinias at higher concentrations.The “tea bag” treatment killed the Salvinias bottom-up by damaging theroot-like submerged leaves of giant Salvinia first (FIG. 5). Then thetrichomes on the upper surfaces of the floating leaves of giant Salviniawere wilted following the damage of the submerged leaves (FIG. 6). Thewilted trichomes released autotoxic compounds and thus enhanced theendocidal function. The “bottom-up” killing style of the “tea bag” isparticularly effective in a shallow and still water body infested withmulti-layers of Salvinia mat because the dead tissues of the bottomlayer of Salvinias killed by the “tea bag” would enhance the endocidalfunction to eliminate the next layer of the Salvinia.

In some cases, because higher effective concentration of dried groundplant Salvinia is needed to inhibit giant Salvinia, and some naturalfactors in lakes and other water systems may dilute the concentrationsor prohibit the occurrence of higher concentrations, it may be necessaryto place dried and ground matter of giant Salvinia in nylon or other netbags to have effective concentrations for the controlling target. Inanother cases, it was found that unground raw plant matter of giantSalvinia at 16 g/L was less effective than ground matter and took morethan five times longer to kill the plants.

To identify more effective agents from giant Salvinia to control thegrowth of the plant, experiments were conducted with a particular focuson water extracts of giant Salvinia (FIG. 1). It was confirmed thatwater extract of giant Salvinia can successfully eliminate giantSalvinia within 13 weeks at RT and prohibit the plant growth for atleast eight months (FIG. 7), MeOH/CH₂Cl₂ and MeOH fractions eliminatedall giant Salvinia plants within two weeks (FIG. 8), new compoundsalviniside II also eliminated the giant Salvinia plants (FIGS. 9-11),and the ethanol extract of giant Salvinia inhibited the growth of giantSalvinia effectively. In summary, while the bioactive MeOH/CH₂Cl₂ andMeOH fractions and salviniside II compound are more effective, eachextract or fraction thereof can be produced as endocides to eliminateand prohibit giant Salvinia.

The experiments showed that extraction temperature and solvent (water)volume significantly contributed to the quality of water extracts andits fractions. The higher extraction temperature (60° C.) and higherrate of solvent volume to dried plant matter weight (43.8:1, v/w)yielded an extraction rate of approximately 8.8% (Example 5), and thelower temperature (RT) and lower rate of solvent volume to dried plantmatter weight (17.2:1, v/w) yielded a 6.25% extraction rate (Example 3).Also, H₂O fraction from the Example 5 (FIG. 14) was much more effectivethan that from Example 3 (FIG. 8).

The experiments indicate that extracts (juice) expressed from the freshmatter of giant Salvinia (e.g., by hydraulic press) can be directly usedin control of giant Salvinia (FIGS. 16-20). The yield rate of extractedjuice from the fresh matter of giant Salvinia may depend on the growthstage and moisture content of the harvested plant matter andapproximately 50% of the plant matter in fresh weight. The extractsexpressed from the fresh matter of giant Salvinia (0.28% or 0.40% inconcentration) were even more effective than the concentrated waterextracts (5%) from the dried matter of giant Salvinia in inhibition ofthe species (FIG. 20). The HPLC analysis showed that the hydraulicextracts had more complex chemical constituents in both detectionconditions and higher contents of some compounds and even uniquecompounds may be responsible for the strong bioactivity of the hydraulicextract (FIGS. 21 and 22). Like aquatic herbicides, addition of asurfactant to the endocide may help it wet and penetrate the floatingleaves with water-repellent trichomes and thereby result in moreeffective spray applications (FIGS. 14, 16, 17, and 20). In bothexperiments, all plant tissues of giant Salvinia after hydraulicextraction were dead and there was no new plant growth in the culturedplant residues from the hydraulic extraction within six weeks of theexperiment (FIG. 19). The plant residues from the extraction of thefresh matter are safe to be discarded into the treated water bodies.Therefore, extracts from the fresh matter of giant Salvinia aredesirable for use as endocides to eliminate and prohibit giant Salvinia.In its concentrated form, the extract juice from fresh matter may alsobe effective to control Salvinia. Because the degraded hydraulic extractafter long-storage might be still useful in control of Salvinias, it isspeculated that diluted several times with water, the newly producedextract juice from the fresh matter may also be effective to controlSalvinias.

According to the experiments, aqueous products of endocides may bebetter to be stored under refrigeration (4° C.) while powder samples canbe stored at RT for relative stability. In general, the H₂O fraction wasmore stable than the water extract or the hydraulic extract in storage.The extract powder stored at RT for 26 weeks had approximately 70% ofthe total peak area of that in the newly prepared extract (FIG. 12).

The hydraulic extract stored under refrigeration (4° C.) is morestabilized than that stored at RT, and on the 14th week of the storageit contained approximately 60% of the total peak area of that in thenewly-prepared extract, and it was still active in inhibition of giantSalvinia (FIGS. 23 and 24). After being stored at RT for 26 weeks, theH₂O fraction powder preserved over 90% of its chemical contents, and itsliquid form stored in a refrigerator (4° C.) still had approximately 90%of the total peak area of the newly prepared sample (FIG. 13).

Salvinia spp. have the potential to hyperacumulate a wide range of heavymetals from water and have high levels of tolerance to the metalsaccording to the literature. The ICP analysis showed that theconcentrations of the main metals in the water extract powder are muchlower than the toxic concentrations in the metal solutions to growSalvinia spp. as reported (10 to 100 ppm). For example, theconcentrations of cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu),lead (Pb), and molybdenum (Mo) in the dry extract powder are less than 1ppm in dry weight, and the concentration of copper (Cu), iron (Fe), lead(Pb), and zinc (Zn) in the dry extract powder is 5.310525, 228.0387,2.515505, and 166.7952, respectively (FIG. 25). The concentrations ofthese metals in the application of endocides were diluted by 20 to 1,000times when using water extract at its spray concentration (0.1 to 5% indry weight) in control of Salvinia. Thus, toxic metals are not the majoractive ingredients of Salvinia endocides, i.e., they are not responsiblefor inhibiting the survival and growth of giant Salvinia.

The experiment showed that isolated salts account for 6.25% of the waterextract from the dried matter of giant Salvinia in dry weight. Of the 5%water extract (the highest concentration used in the experiments), theconcentration of the salts is 0.3125% (3.125 ppt). The experimentindicated that even the spray of the salts at even 5% concentration (50ppt) of the salts had no significant impact on the growth of giantSalvinia. Therefore, the salts precipitated from the water extract arenot the major active ingredients responsible for the bioactivity ofwater extracts (FIG. 25).

The experiments indicated that both spraying and dissolving these agentswere effective. A similar approach may be taken to identify endocides inall invasive plant species, including but not limited to Salviniaadnata, S. auriculata, S. biloba, S. cucullata, S. cyathiformis, S.hastata, S. herzogii, S. martynii, S. minima, S. molesta, S. natans, S.nymphellula, S. oblongifolia, S. radula, S. rotundifolia, S. sprucei,Azolla caroliniana, A. cristata, A. circinata, A. filiculoides, A.japonica, A mexicana, A. microphylla, A. nilotica, A. pinnata, A. rubra,Lemna minuta, or Eichharnia crassipes.

The water extract of dried matter of giant Salvinia can totallyeliminate the plants of the floating fern (S. minima), a species withinthe same genus with giant Salvinia, at either 0.1% or 0.5% within sixweeks (FIG. 26). Both water extracts and hydraulic extracts of giantSalvinia had significant impacts on the growth of Carolina mosquito fern(Azolla caroliniana), a fern species in a different family but the sameorder with the genus Salvinia. The hydraulic extract (0.4%) killed 100%of the Carolina mosquito fern plants by the end of the 15th day (FIG.27). The water extract at higher concentrations (2.5, 5, or 7.5%) killed100% of the Carolina mosquito fern plants within the 15 days ofexperiment (FIG. 27). At even 5% concentration, the water extract of thedried matter of giant Salvinia did not impact the growth of herbaceousflowering plants (e.g., least duckweed (Lemna minuta), Brazillanwatermeal (Wolffia brasillensis Weddell), water hyacinth (Eichharniacrassipes), and hydrilla (Hydrilla verticillata (L.f.) Royle)), andnative confers (e.g., bald cypress (Taxodium distichum (L.) Rich.) andloblolly pine (Pinus taeda L.)). The endocide of giant Salvinia hasobvious selective activity against Salvinias (order Salviniales)especially the species of family Salviniaceae, but it has no impacts onseed plants (spermatophytes) including angiosperms (flowering plants)and gymnosperms.

2. Natural Pesticides Derived from Second Species

Least duckweed (Lemna minuta) is a noxious invasive species itself. Itswater extract successfully eliminated giant Salvinia and prohibitedplant growth for at least eight months (see Example 14) (FIGS. 28 and29). Ethanol extract of duckweed was also effective to control giantSalvinia (FIG. 30). Duckweed extracts were also able to control Salviniaminima (FIGS. 31-33).

B. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Elimination and Prohibition of Giant Salvinia (SalviniaMolesta) by the Water-Extracted Dried Matters of Giant Salvinia

General Experimental Procedures:

Air-dried whole plants of giant Salvinia were ground to a coarse powderand placed in nylon net bags (called “tea bags”). Each of 12 containershad 400 g of living healthy plants of giant Salvinia in all growthstages (primary, secondary, and tertiary stages) in 25 L of tap water inthe greenhouse (30° C. during the day time and 20° C. at night). Threecontainers had no treatment to serve as controls. The first treatmentgroup had three containers and each had one bag of 100 g of giantSalvinia dried matter in the water. The second group of three containershad one bag of 200 g of giant Salvinia dried matter, and the last groupof three containers had one bag of 400 g of giant Salvinia dried matter.There was no significant difference of pH values between the treatmentsand control during the experiments.

Results:

In the 400 g treatment group, more than 35% of plants were dead by theend of the first week and all plants were dead by the end of the secondweek and no plants survived or new growth occurred thereafter during theobservation of six months (FIGS. 3 and 4). In this treatment, theroot-like submerged leaves of giant Salvinia were totally damaged abouta week later and then the floating leaves turned into brown and dead(FIG. 5). In contrast to those in the normal plant growth condition, thetrichomes on the upper surfaces of the floating leaves of giant Salviniawere soon wilted following the response of submerged leaves to the “teabag” treatment (FIG. 6). The wilted trichomes released autotoxiccompounds and thus enhanced the endocidal function. It was also foundthat the 200 g treatment group inhibited almost 50% of the plants by theend of the fourth week of the experiment. However, no significant effectof the 100 g treatment was observed on Salvinia growth.

Example 2 Elimination and Prohibition of Giant Salvinia Plants by WaterExtract of the Dried Matter of Giant Salvinia

General Experimental Procedures:

Air-dried whole plants (350 g) were ground to a coarse powder andpercolated with H₂O at RT to yield a 4 L aqueous solution (0.84%, g/mL).Of the aqueous solution, 119 mL was diluted to 900 mL 0.1% and 1,000 mL0.01% solution (g/mL) for the following experiment. The experimentincluded 45 healthy and untreated living plants of giant Salvinia (insecondary growth stage, approximately 7 g in fresh weight each). Theplants were cultured and tested in plastic containers (14×15 cm, 0.68 L)in the NCPC Lab at RT. Controls: 15 plants with five in each container(3 replications) were cultured with 300 mL of tap water; 0.01% waterextract treatment: 15 plants with five in each container (3replications) were sprayed with 300 mL 0.01% of the water extract ofgiant Salvinia dissolved in tap water; and 0.1% water extract treatment:15 plants with five in each container (3 replications) were sprayed with300 mL 0.1% of the water extracts of giant Salvinia dissolved in tapwater. Plant growth and survival status were documented and photographedweekly after the treatments. By the end of the 13th week, new growthbiomass of the plants in each treatment was measured. The biomass of newgrowth was a primary factor to measure the inhibition of each treatmenton the target plant.

Results:

By the end of 13 weeks of treatment, each of the 15 plants in thecontrol group had new growth, and 12 of the total 15 plants treated with0.01% water extract of giant Salvinia had new growth (FIG. 7). All giantSalvinia plants treated with 0.1% water extract of giant Salvinia weredead and no new growth developed from any of the plants during the eightmonths of observation. pH values of the culture solution varied from 7.4to 7.6 and there were no significant differences among control andtreatments.

Example 3 Elimination and Prohibition of Giant Salvinia Plants byFractions of Water Extract of the Dried Matter of Giant Salvinia

General Experimental Procedures:

Air-dried whole plants of giant Salvinia (1.4 kg) were ground to acoarse powder and extracted two times for 48 h with H₂O (12 L×2) at RT.The combined H₂O extracts were concentrated to give extracts (88 g)under reduced pressure. The H₂O extracts was applied on a column ofsilica gel (1,000 g) eluting with the mixture of MeOH/CH₂Cl₂ (1:1, v/v,3 L), 3 L of 100% MeOH, and 1 L H₂O to obtain three fractions,respectively (FIG. 1). The water extract and three fractions were allprepared as experimental solutions at the concentration of 0.1%. Thetotal 45 healthy and untreated living plants of giant Salvinia (insecondary growth stage, approximately 10 g in fresh weight each) werecultured and tested in the plastic containers (14×15 cm, 0.68 L) in theNCPC Lab at RT. The five treatments are as follows. Controls: 9 plantswith three in each container (3 replications) were cultured with 150 mLof tap water; water extract treatment: 9 plants with three in eachcontainer (3 replications) were sprayed with 150 mL 0.1% of the waterextract of giant Salvinia dissolved in tap water; H₂O fractiontreatment: 9 plants with three in each container (3 replications) weresprayed with 150 mL 0.1% of H₂O fraction dissolved in tap water; MeOHfraction treatment: 9 plants with three in each container (3replications) were sprayed with 150 mL 0.1% of MeOH fraction dissolvedin tap water; and MeOH/CH₂Cl₂ fraction treatment: 9 plants with three ineach container (3 replications) were sprayed with 150 mL 0.1% ofMeOH/CH₂Cl₂ (1:1) fraction dissolved in tap water. Plant growth andsurvival status were documented and photographed weekly after thetreatments. By the end of 2nd week, new growth biomass of plants in eachtreatment was measured. The biomass of new growth was a primary factorto measure the inhibition of each treatment on the target plant.

Results:

On the 3rd day after the treatment, all plants treated with MeOH/CH₂Cl₂or MeOH fraction were dead in comparison to 100% survival of the plantsin the control group. During the same day of observation, some leaves ofthe plants treated with water extract or H₂O fraction started to turninto brown color. By the end of two weeks, all plants in the controlgroup were still alive (FIG. 8). By then, in contrast, no plantssurvived or new growth in both MeOH/CH₂Cl₂ and MeOH treatments, almostall plants were dead with water extract, while the biomass of livingplants under the H₂O fraction treatment was less than 25% of the plantsin the control group (FIG. 8). The experiment indicated that MeOH/CH₂Cl₂and MeOH fractions were very active and can fully control giant Salviniamore quickly than its parent water extract and thus the followingisolation of bioactive compounds was focused on these two fractions.

Example 4 Elimination and Prohibition of Giant Salvinia by SalvinisideII, an Isolate from Water Extract of the Dried Matter of Giant Salvinia

General Experimental Procedures:

Continuous fractionation was based on the combined MeOH/CH₂Cl₂ (1:1,v/v) and 100% MeOH fractions which showed potent activity to inhibitgrowth of giant Salvinia in Example 3. The combined fractions wereloaded on a pre-equilibrated open ODS column (60×600 mm). The ODS columnwas eluted successively with 30%, 55%, 75% and 100% MeOH to yield fourfractions F1, F2, F3, and F4, respectively. F3 (75% MeOH elute) wasconcentrated and separated by a preparative HPLC (MeOH/H₂O, 35:65, 254nm) to yield four compounds including active salviniside II and C3 andinactive C1 and C2 and active fraction F32 and inactive F31 (FIG. 1).NMR experiments were performed on a JEOL ECS-400 and a Bruker Avance 700NMR instrument. NMR data were reported as δ (ppm) values and referencedto the solvent used. HRESIMS were acquired on an electrospray instrument(MDS Sciex Pulsar Qstar, Ontario, Canada). Octadecyl-functionalizedsilica gel, silica gel, Sephadex LH-20, and TLC plates were purchasedfrom Aldrich Chemical Co. HPLC analysis was performed on a HewlettPackard Series 1100 with a HP 1100 diode array detector using a HypersilODS column (150×4.6 mm, 5 μM, Supelco; flow rate, 1 mL/min; MeOH/H₂O(v/v) linear gradient, 2:98-98:2 in 35 min). Preparative HPLC wasperformed with an Acuflow Series III pump connected with an Acutect 500UV/VIS detector using an Econosil ODS column (250×22 mm, 10 μM, Alltech). The total 27 healthy and untreated living plants of giantSalvinia (in secondary growth stage, approximately 10 g in fresh weighteach) were cultured and tested in plastic containers (14×15 cm, 0.68 L)in the NCPC Lab at RT. The five treatments were as follows. Controls: 9plants with three in each container (3 replications) were cultured with150 mL of tap water; 0.01% salviniside II treatment: 9 plants with threein each container (3 replications) were sprayed with 150 mL 0.01% ofsalviniside II dissolved in tap water; and 0.1% salviniside IItreatment: 9 plants with three in each container (3 replications) weresprayed with 150 mL 0.1% of salviniside II dissolved in tap water. Plantgrowth and survival status were documented and photographed weekly afterthe treatments. By the end of two weeks, new growth biomass of plants ineach treatment was measured. The biomass of new growth was a primaryfactor to measure the inhibition of each treatment on the target plant.

Results:

The growth of giant Salvinia was totally inhibited by salviniside II(FIGS. 9 and 10) at 0.1% of concentration within two weeks in the lab atRT (FIG. 11). There was no new growth identified thereafter during theeight months of observation.

Salviniside II is a new compound. It was obtained as colorless powdersand showed an [M-1]⁺ at m/z 473.0714 (calcd for 473.0720) in theHRESIMS, suggesting the molecular formula to be C₂₂H₁₈O₁₂. The ¹³C and¹H-NMR spectrum (FIG. 10) displayed similarly two set of glycosidesignals with two anomeric proton at δ 4.57 (1H, d, J=7.8 Hz, β-H-1″) andδ 5.14 (1H, d, J=3.7 Hz, α-H-1″), and two protonated carbon at δ_(C-β)98.6 and δ_(C-α) 94.4 observed in the HSQC spectrum. The analysis of theCOSY, HSQC spectra of salviniside II indicated it is an isomeric mixtureof α and β-D-glucopyranosides (Choudhary et al. 2008; Ding et al. 1999;Narasimhulua et al. 2010). In addition, each signals of the aglycon partappeared essentially in duplicate owing to the formation of a mixture ofα and β-anomers in the NMR spectrum. Five aromatic protons were found inthe ¹H-NMR spectrum (FIG. 10), of which three signals at δ 7.39 (1H, dd,J=1.8 and 8.5 Hz, H-2′), δ 6.84 (1H, d, J=8.5 Hz, H-3′), and 7.45 (1H,d, J=1.8 Hz, H-6′) were characteristic of a 1,2,4-trisubstituted phenylmoiety, the remaining two at δ 7.70 (1H, d, J=8.2 Hz, H-6) and 6.82 (1H,d, J=8.2 Hz, H-7) were assigned to a 1,2,3,4-tetra substituted phenylgroup (Narasimhulua et al. 2010). A close inspection of the ¹³C-NMRspectrum data, together with the MS data, clearly revealed that the1,2,3,4-tetrasubstituted phenyl moiety should be a part of a 8-carbonbenzofuran skeleton (Maurya et al. 2004; Wu et al. 2011). The ¹³C NMRspectra of salviniside II displayed two ester carbonyl carbons at δ168.1 and δ 168.7. As observed in the HMBC spectrum, two key HMBCcorrelation among H-6 at δ_(H) 7.70 and Glc H-6″ at δ_(H) 3.94 with acarbonyl carbon at δ_(C) 168.1 (C-11) established the linkage betweenthe benzofuran unit and H-6″ of Glc. Similarly, the other ester carbonylcarbon (δ_(C) 168.7, C-10) was attached to H-4″ of Glc position by theHMBC spectrum analysis. These long-range HMBC correlations of H-2′ toC-1′, C-6′ and C-2, and of H-6′ to C-1′, C-2′ and C-2 established theconnectivity of the benzofuran skeleton with 1,2,4-trisubstituted phenylmoiety. The 1,1-ADEQUATE spectrum also confirmed the assignment. Therelatively downfield shifted of C-3 at δ 110.3 and the remaining onedegree of unsaturation established the linkage of the C-3 and C-10. Thecomplete assignment of the protons and carbons was achieved by acombination of ¹H, ¹³C, HSQC, ¹H-¹H COSY, HMBC and 1,1-ADEQUATE spectralanalyses. Thus, salviniside II was identified as4″,6″-O-[3,5-dicarbonyl-8-hydroxy-2-(4′,5′-dihydroxy-phenyl)-1-benzofuran-2-yl]-D-glucopyranose.

Example 5 Elimination and Prohibition of Giant Salvinia Plants by WaterExtract and H₂O Fraction of the Dried Matter of Giant Salvinia withSurfactants

General Experimental Procedures:

Air-dried whole plants of giant Salvinia (8 kg) were ground to a coarsepowder and extracted in a vacuum distillation system (Eden Labs,Seattle, USA) with H₂O two times for 24 h each (200 L and 150 L,respectively) at 60° C. The combined H₂O extracts were concentrated togive an extract (703 g) under reduced pressure. The water extract wasmixed with 300 g silica gel and loaded into a column of silica gel(1,000 g). The column was then eluted with 4 L of MeOH and 4 L H₂O. Thewater extract and H₂O fraction were both prepared as experimentalsolutions at the concentration of 5%.

The HPLC chromatographs of the newly prepared water extract and H₂Ofraction were also established by Agilent 1100 HPLC system coupled to anAgilent 1100 diode array detector, and an Eclipse XDB-C18 column(4.6×150 mm, 3.5 μM) at a flow rate of 0.6 mL/min. A gradient elutionwas performed by using H₂O (A) and CH₃CN (B) as mobile phases. Elutionwas performed according to the following conditions: 2% B at time 0,linear increase to 98% B in 22 min, and hold 98% B for 8 min. The columntemperature was maintained at 23° C. The HPLC chromatogram wasstandardized on retention times and peak intensities of the peaksobserved at a wavelength of 254 nm. The concentration of water extractwas 24 kg fresh plant matter/L and the injection volume was 3 μL for allanalyses. The concentration of H₂O fraction was 16 kg fresh plantmatter/L. The injection volume was 50 μL for all analyses.

Part of each of water extract and H₂O fraction was prepared separatelyas dry powder by SPD 2010 Integrated SpeedVac (Thermo Scientific, NC,USA) for 12 h and then was stored at RT for 26 weeks. In addition, both5% water extract and 5% H₂O fraction were stored at refrigerator (4° C.)for 26 weeks. To determine the stability of the water extract and H₂Ofraction during the storage, HPLC profiles of the powder and aqueoussamples were established by Agilent 1100 at the condition describedabove.

The experiment included 18 containers and each container had 750 g (infresh weight) of healthy living Salvinia molesta in all growth stages(primary, secondary, and tertiary stages) cultured in 57 L of rainwater. The plants were cultured and tested in the glass tank in the labat RT. There were six treatments. Controls: each of the three containershad 750 g giant Salvinia plants cultured with rain water; “tea bag”treatment: each of the three containers with a nylon net bags of 800 gground air-dried plant matter of giant Salvinia on the bottom (seeExample 1); water extract treatment: plants in each of the threecontainers were sprayed twice (on the 1^(st) and 18^(th) day), each timewith 100 mL 5% of the water extract of giant Salvinia dissolved inwater; water extract with surfactants treatment: plants in each of thethree containers were sprayed twice (on the 1^(st) and 18^(th) day),each time with 100 mL 5% of the water extract of giant Salviniadissolved in water with 0.5 mL Inlet™ (polyalkoxylated andnon-alkoxylated aliphatics and derivatives 90%, Helena Chemical Company,Collierville, Tenn., USA) and 0.25 mL Kinetin® (proprietary blend ofpolyalkyleneoxide modified polydimethylsiloxane 99%, Helena ChemicalCompany, Collierville, Tenn., USA); H₂O fraction treatment: plants ineach of the three containers were sprayed twice (on the 1^(st) and18^(th) day), each time with 100 mL 5% of H₂O fraction of the waterextract of giant Salvinia dissolved in water; and H₂O fraction withsurfactants treatment: plants in each of the three containers weresprayed twice (on the 1^(st) and 18^(th) day), each time with 100 mL 5%of H₂O fraction of the water extract of giant Salvinia dissolved inwater with 0.5 mL Inlet™ and 0.25 mL Kinetin®. Plant growth and survivalstatus were documented and photographed weekly after the treatments. Bythe end of 3^(rd) or 4^(th) week, biomass of living plants in eachtreatment was measured.

Results:

According to HPLC analyses, the powder samples stored at RT for 26 weekswere relatively stable. By the end of 26 weeks, the extract powder stillhad approximately 70% of the total peak area of that in the newlyprepared extract (FIG. 12). By the end of 26 weeks, H₂O fraction powderstored at RT preserved over 90% of its chemical contents and 5% H₂Ofraction liquid stored at refrigerator (4° C.) still had approximately90% of the total peak area of the newly prepared sample (FIG. 13).

The growth of giant Salvinia can be successfully controlled by theendocide treatments in comparison with control (FIG. 14). By the end ofthe third week, all plants treated with either “tea bag” (FIG. 15),water extract with surfactants, or H₂O fraction of the water extractwith surfactants were dead and there was no new growth during thefollowing four months of observation. By the end of 4^(th) week, over90% or 95% of the plants treated with water extract and H₂O fraction ofthe water extract were dead, respectively. Obviously, Surfactants madethe Salvinia endocides more effective. Because the experiments wereconducted in November and at RT, plants experienced relatively slowgrowth. The living biomass in the controls had lightly decreased becausethe mature tissues were dead and more young plants emerged during thefour weeks of experiment.

Example 6 Elimination and Prohibition of Giant Salvinia Plants byHydraulic Extract of the Fresh Matter of Giant Salvinia

General Experimental Procedures:

The total 58 kg fresh matter of giant Salvinia plants were chopped andshredded by food processor. Then the processed material was pressed byhydraulic press (Enerpac) at pressure of 27,579 kPa (4,000 psi) for 2min to get 30 kg extract juice. To obtain the concentration of theextract juice, three 30 mL of juice samples were randomly collected andmeasured. Each of the three juice samples was centrifuged by accuSpin 3RBenchtop centrifuge (Fisher Scientific, PA, USA) at 3,000 rpm for 10min. The upper liquid was first dried by SPD 2010 Integrated SpeedVac(Thermo Scientific, NC, USA) for 12 h. Then dried extract was measured.The average result of the three test samples was used to estimate theconcentration of the extract juice. The concentration of hydraulicextract juices were 2 kg fresh plant matter/L. The spray treatmentexperiment included three plastic containers (20 gal, approximatelyequal to 75.7 L) with approximately 390 g (in fresh weight) of giantSalvinia living plants (in all growth stages: primary, secondary, andtertiary stages) in 50 L of tap water each in the greenhouse (30° C.during the day time and 20° C. at night). There were three treatments.Control: the plants in the first container were sprayed with 150 mL tapwater only; extract juice treatment: the plants in the second containerwere sprayed with 150 mL of extract juice; and extract juice andsurfactants treatment: the plants in the third container were sprayedwith 150 mL extract juice with 0.375 mL Inlet™ (polyalkoxylated andnon-alkoxylated aliphatics and derivatives 90%, Helena Chemical Company,Collierville, Tenn., USA) and 0.1875 mL Kinetin® (proprietary blend ofpolyalkyleneoxide modified polydimethylsiloxane 99%, Helena ChemicalCompany, Collierville, Tenn., USA). The second same spay treatment wasapplied to each of these six contains a week later, respectively. Plantgrowth and survival status were documented and photographed weekly afterthe treatments. By the end of 6th week, the total biomass of livingplants in each treatment was measured. To investigate the survival rateof giant Salvinia after hydraulic extraction, the 28 kg (in freshweight) residual plant material of giant Salvinia expressed by hydraulicpress was cultured in five containers with 50 L tap water each for sixweeks.

Results:

The extraction rate of hydraulic extract from the fresh matter of giantSalvinia in this experiment was approximately 51.7%. Based on the threetest samples, the estimated concentration of the experimental extractjuice was 0.28% (in fresh weight). The spray experiment showed that thehydraulic extract juice effectively inhibited the giant Salvinia aftertwo treatments (FIGS. 16 and 17). In comparison with those in thecontrol treatment, the trichomes on the upper surfaces of the floatingleaves of giant Salvinia treated by the hydraulic extract hadsignificant damage (FIG. 18). The wilted trichomes released autotoxicchemicals to enhance the endocidal function of the hydraulic extract.With surfactants, hydraulic extract juice totally eliminated the giantSalvinia within four weeks and no new growth thereafter. The experimentindicates that the juice expressed from fresh matter of giant Salvinia(e.g., by hydraulic press) is effective enough and can be directly usedin control of giant Salvinia without concentrating process. All planttissues of giant Salvinia after hydraulic extraction were dead and therewas no new plant growth in the cultured plant residues from thehydraulic extraction within six weeks of observation (FIG. 19).

Example 7 Elimination and Prohibition of Giant Salvinia Plants by WaterExtract from the Dried Matter and Hydraulic Extract of the Fresh Matterof Giant Salvinia with Surfactants

General Experimental Procedures:

The 5% water extract from the dried matter of giant Salvinia (called“water extract”) was prepared as described in the Example 5. The total80 kg fresh matter of giant Salvinia plants were chopped and shredded bychipper shredder CS 3310 (Cub Cadet, Cleveland, Ohio, USA). Then theprocessed material was pressed by hydraulic press (Enerpac) at pressureof 27,579 kPa (4,000 psi) for 2 min to get 38 kg extract juice. Toobtain the concentration of the hydraulic extract, three 30 mL of juicesamples were randomly collected and measured. Each of the three juicesamples was centrifuged by accuSpin 3R Benchtop centrifuge (FisherScientific, PA, USA) at 3,000 rpm for 10 min. The upper liquid was firstdried by SPD 2010 Integrated SpeedVac (Thermo Scientific, NC, USA) for12 h. Then dried extract was measured. The average result of the threetest samples was used to estimate the concentration of the hydraulicextract (called “hydraulic extract”). The HPLC profiles of both waterextract and hydraulic extract were established before the firsttreatments by using both of the following methods. (1) Method A: Agilent1100 HPLC system coupled to an Agilent 1100 diode array detector, and anEclipse XDB-C18 column (4.6×150 mm, 3.5 μM) at a flow rate of 0.6mL/min. A gradient elution was performed by using H₂O (A) and CH₃CN (B)as mobile phases. Elution was performed according to the followingconditions: 2% B at time 0, linear increase to 98% B in 22 min, and hold98% B for 8 min. The column temperature was maintained at 23° C. TheHPLC chromatogram was standardized on retention times and peakintensities of the peaks observed at a wavelength of 254 nm. Theconcentration of both water extract and hydraulic extract were 2 kgfresh plant matter/L. The injection volume was 50 μL for all analyses.(2) Method B: Agilent 1260 HPLC system coupled to an Agilent 1260 diodearray detector, and an Acclaim HILIC-10 column (4.6×150 mm, 3.0 μM) at aflow rate of 0.6 mL/min. The mobile phase was CH₃CN/H₂O (65:35, v/v).The column temperature was maintained at 23° C. The HPLC chromatogramwas standardized on retention times and peak intensities of the peaksobserved at a wavelength of 230 nm. The concentration of both waterextract and hydraulic extract were 40 kg fresh plant matter/L. Theinjection volume was 20 μL for all analyses. To determine the stabilityof hydraulic extract, HPLC profiles of the hydraulic extracts stored atrefrigerator (4° C.) and RT was established separately by above Method Adaily for the first week and on the 14^(th) week before the thirdtreatment in comparison with the profiles of the newly prepared extractfor the first treatments. The spray treatment experiment included six625 gal (approximately 2,366 L) containers with approximately 7,400 g(including approximately 7,000 g of plants in tertiary growth stage,approximately 300 g of plants in secondary growth stage, andapproximately 100 g of plants in primary growth stage) of giant Salvinialiving plant in 1,700 L of rain water each in the greenhouse (30° C.).There were three treatments. Control: the plants in each of the twocontainers were sprayed with 2.5 L rain water only; water extract withsurfactants treatment: the plants in each of the two containers weresprayed with 2.5 L of 5% water extract with 6.25 mL Dyne-Amic® (methylesters of C16-C18 fatty acids, polyalkyleneoxide modified polydimethylsiloxane, alkylphenol ethoxylate 99%, Helena Chemical Company),and 3.125 mL Kinetin® (proprietary blend of polyalkyleneoxide modifiedpolydimethylsiloxane 99%, Helena Chemical Company, Collierville, Tenn.,USA); and hydraulic extract and surfactants treatment: the plants ineach of two containers were sprayed with 2.5 L extract juice with 6.25mL Dyne-Armic®, and 3.125 mL Kinetin®. The second spay treatment at thesame dosage was applied to each of these six contains 10 days later,respectively. Plant growth and survival status were documented andphotographed weekly after the treatments. By the end of 14th week, thetotal biomass of living plants in each treatment was measured. Then thethird spay treatment at the dosage of 0.5 L (for water extract andextract juice, each with 1.25 mL Dyne-Amic®, and 0.625 mL Kinetin® wasapplied to each of these six contains, respectively. To investigate thesurvival rate of giant Salvinia after hydraulic extraction, the 42 kg(in fresh weight) residual plant material of giant Salvinia expressed byhydraulic press was cultured in six containers with 50 L tap water eachfor six weeks.

Results:

The extraction rate of hydraulic extract from the fresh matter of giantSalvinia in this experiment was approximately was 47.5%. Based on thethree test samples in each experiment, the estimated concentration ofthe experimental hydraulic extract was 0.40% (in fresh weight). Thehydraulic extract in this experiment had lower extraction rate of yieldat higher concentration than that in the Example 6 because the plantmatter used in this extraction had more dead tissues which holding lessmoisture. This experiment showed that both water extract and hydraulicextract with surfactants effectively inhibited the giant Salvinia aftertwo treatments (FIG. 20). The hydraulic extract with surfactants totallyeliminated the giant Salvinia within 15 weeks. The experiment indicatesthat hydraulic extract expressed from the fresh matter of giant Salviniawas effective enough without concentrating process and can be useddirectly in inhibition of the species (FIG. 20). All plant tissues ofgiant Salvinia after hydraulic extraction were dead and there was no newplant growth in the cultured plant residues from the hydraulicextraction within six weeks of observation.

The experiment indicates that hydraulic extract expressed from the freshmatter of giant Salvinia at low concentration (0.40%) was more effectivethan the concentrated water extract (5%) from the dried matter of giantSalvinia in inhibition of giant Salvinia. HPLC profiles indicate thathydraulic extracts had more complex chemical constituents in bothdetection conditions and higher contents of some compounds and evenunique compounds may be responsible for the strong bioactivity of thehydraulic extract (FIGS. 21 and 22).

According to HPLC analyses, hydraulic extract degraded much more quicklyat RT than that at refrigerator (4° C.). The total peak area ofhydraulic extract stored at RT decreased to 35% by the end of the firstweek and to 30% by the end of the second week (vs. 70% and 66% stored atrefrigerator (4° C.), respectively) in comparison with the newlyprepared extract (FIG. 23). On the 14th week, hydraulic extract storedat refrigerator (4° C.) still contained approximately 60% of the totalpeak area of that in the newly prepared extract (first treatment) andstill was active in inhibition of giant Salvinia during the third spraytreatment (FIG. 24).

Example 8 Elimination and Prohibition of Giant Salvinia Plants byHydraulic Extract of the Fresh Matter of Giant Salvinia in Combinationwith Formic Acid or Acetic Acid

General Experimental Procedures:

Formic acid (90.4%, certified ACS reagent grade, Fisher ScientificCompany, Fair Lawn, N.J., USA) and acetic acid (99.7, ACS reagent grade,VWR International LLC, West Chester, Pa., USA) were prepared assolutions at the concentration of 0.01%, 0.1%, 1%, 5%, and 10%immediately before the bioassay experiments. The experimental hydraulicextract used in this example was prepared from the previous experiment(fresh matter of giant Salvinia (method see Example 7). The actualconcentration of the extract during this experiment was approximately0.28%. There were 37 treatments in this experiment. Each treatmentincluded 9 living healthy plants of giant Salvinia in three Rubbermaidplastic containers (14×15 cm, 0.68 L) (each container had two plants insecondary stage and one in tertiary growth stage, in total of 6 g infresh weight). The plants in each container were sprayed with 12.5 mL oftap water, formic acid, acetic acid, or combination of hydraulic extractwith formic or acetic acid. Control: the plants was sprayed with wateronly; nine formic acid treatments (0.01%, 0.05%, 0.1%, 0.25%, 0.5%,0.75%, 1%, 5%, and 10% concentrations); nine acetic acid treatments(0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 5%, and 10%concentrations); nine hydraulic extract treatments with variousconcentrations of formic acid (0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%,1%, 5%, and 10%); and nine hydraulic extract treatments with variousconcentrations of acetic acid (0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%,1%, 5%, and 10%). Plant growth and survival status were documented andphotographed hourly for the first 5 h and then daily for seven daysafter the treatment.

Results:

Giant Salvinia is very sensitive to both formic acid and acetic acid.All plants treated with either formic or acetic acid at the 1% or higherconcentrations were dead within 3 h of the treatment. However, at lowerconcentrations (<1%), formic acid was much more effective than aceticacid in control of giant Salvinia. For example, 0.05% formic acid wasmore effective than 0.25% acetic acid in giant Salvinia. Formic acid atthe 0.01% concentration still effectively inhibited the growth of giantSalvinia. By combing hydraulic extract with 0.25% formic acid, all giantSalvinia plants were killed within 24 h. Treated by combing hydraulicextract with 0.05% formic acid, all giant Salvinia plants were deadwithin a week. Treated by combing hydraulic extract with 0.01% formicacid, more than 40% of the giant Salvinia plants were dead during thefirst week. The Salvinia plants responded to the hydraulic extract withthe organic acids much quickly and severely than hydraulic extract,formic acid, or acetic acid alone.

Example 9 Metal Analysis of the Dried Matter of Giant Salvinia and itsWater Extract and Extract Salts

General Experimental Procedures:

The dried matter of giant Salvinia and its water extract used thisexperiment were from Example 3. From the MeOH fraction of the 88 g waterextract of the dried matter of giant Salvinia (see Example 3), 5 g saltswere precipitated. 0.5 g of each of the ground dried plant matter ofgiant Salvinia, water extract powder, and the salts was pre-digestedovernight with 5 mL nitric acid in Digi-tubes at RT. Then the Digi-tubeswere incubated on DigiPREP HT High Temperature Digestion Systems (SCPSCIENCE) for 3 h at 100° C. Cool down and add 4 mL of hydrogen peroxide.Cook for 90 min at 95° C. The solution was diluted to a final volume of50 mL for ICP analysis (Thermo Scientific iCap 7200 ICP-OES). The 5 ppt(0.5% in dry weight) and 50 ppt (5% in dry weight) solutions of thesalts were prepared. 70 g living healthy plants of giant Salvinia intertiary growth stage were cultured in each of nine plastic containers(23×23 cm, 2.4 L). There were three treatments with three containerseach. Control: the plants in each of three containers were sprayed with12.5 mL tap water only; 5 ppt salts treatment: the plants in each of thethree containers were sprayed with 12.5 mL 0.5% salt solutions; 50 pptsalts treatment: the plants in each of the three containers were sprayedwith 12.5 mL 5% salt solutions. Plant growth and survival status weredocumented and photographed weekly for eight weeks after the treatment.

Results:

The ICP analysis showed that the concentrations of the main mealscadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), lead(Pb), molybdenum (Mo), and zinc (Zn), in the dry extract powder were0.16047, 0.883666, not detected, 5.310525, 228.0387, 2.515505, 0.039819,and 166.7952 ppm (in dry weight) (FIG. 25). The salts precipitated fromthe water extract of the dried matter of giant Salvinia were mainlysodium (Na), potassium (K), calcium (Ca), and magnesium (Mg) saltsaccording to the ICP analysis (FIG. 25). The 5 g precipitated saltsaccounts for 0.3333% of the 1.5 kg dry plant matter or 6.25% of the 88 gwater extract in dry weight. Through eight weeks of observation, theplants treated with the salts at either 5 or 50 ppt did not have anysignificant damage. There was no significant biomass difference amongthe salts treatments and control. Obviously, the spray of the salts evenat 5% did not inhibit the plant growth of giant Salvinia. In contrast,water extract of the dry plant of giant Salvinia can effectively controlgiant Salvinia as described in Examples 5 and 6. Thus, the salts werenot the active ingredients of the water extract in responsible forinhibiting giant Salvinia.

Example 10 Elimination and Prohibition of Floating Fern (SalviniaMinima) Plants by Water Extract of the Dried Matter of Giant Salvinia

General Experimental Procedures:

The experiment included nine plastic containers (14×15 cm, 0.68 L) andeach container had 10 g (in fresh weight) of healthy living Salviniaminima cultured in tap water in the greenhouse (30° C. during the daytime and 20° C. at night). There were three treatments. Controls: thefloating fern plants in each of three containers were sprayed threetimes with the total of 90 mL tap water; 0.1% water extract treatment:the plants in each of three containers were sprayed three times with thetotal 90 mL 0.1% of water extract of giant Salvinia (the preparationmethod see Example 2); and 0.5% water extract treatment: the plants ineach of three containers were sprayed three times with the total 90 mL0.5% of water extract of giant Salvinia (the preparation method seeExample 2). Plant growth and survival status were documented andphotographed weekly after the treatments. By the end of 1st, 2nd, 6thweeks, biomass of living plants in each container of all treatments wasmeasured.

Results:

The growth of the floating fern plants treated with either 0.1% or 0.5%water extract of giant Salvinia were obviously inhibited during thefirst week (FIG. 26). By the end of the 6th week, all plants treated bywater extract of giant Salvinia were dead and there was no new growththereafter.

Example 11 Elimination and Prohibition of Carolina Mosquito Fern (Azollacaroliniana) Plants by Water Extract from the Dried Matter and HydraulicExtract of the Fresh Matter of Giant Salvinia

General Experimental Procedures:

The experimental species included a fern species Carolina mosquito fern(Azolla caroliniana) (family Azollaceae). The experiments were conductedin the greenhouse (30° C. during the day time and 20° C. at night).Water extracts were prepared from the dried matter of giant Salvinia(the preparation method see Example 2) and the hydraulic extract wasprepared from the fresh matter of giant Salvinia (see Example 7). Thespray experiment started until the plants fully covered each of the 18plastic containers (14×15 cm, 0.68 L) with 400 mL tap water each. Theplants were randomly classified into six groups: Control: each of thethree containers was spayed three times with 10 mL of tap water eachtime; 0.4% hydraulic extract treatment: each of the three containers wasspayed with 10 mL of 0.4% hydraulic extract; 0.1% water extracttreatment: each of the three containers was spayed twice with 10 mL of0.1% water extract; 2.5% water extract treatment: each of the threecontainers was spayed twice with 10 mL of 2.5% water extract; 5% waterextract treatment: each of the three containers was spayed twice with 10mL of 5% water extract; and 7.5% water extract treatment: each of thethree containers was spayed twice with 10 mL of 7.5% water extract. Bythe end of the 5^(th), 10^(th), and 15^(th) day, surface coverage ofliving plants in each container of all treatments was photographed andmeasured and EC₅₀ (half maximal effective concentration) values werecalculated by the PROBIT procedure of SPSS 13.0 for Windows.

Results:

Neither the hydraulic extract (0.4%) nor the water extract at 0.1%concentration showed any significant impacts on the plant growth ofCarolina mosquito fern during the first five days of the experiment butboth extracts significantly inhibited the plant growth thereafter. Thehydraulic extract was able to kill 100% of the Carolina mosquito fern bythe end of the 15th day (FIG. 27). The water extract at higherconcentrations (2.5, 5, or 7.5%) significantly inhibited the growth ofCarolina mosquito fern soon after the treatments. Both 2.5% and 5% waterextracts killed 100% of the Carolina mosquito fern plants by the end ofthe experiment. The 7.5% water extract eliminated all treated plantswithin the five days of the experiment (FIG. 27). EC50 value of waterextracts of the dried matter of giant Salvinia against Carolina mosquitofern is 4.42%, 1.32%, and 0.16% by the 5^(th), 10^(th), and 15^(th) day,respectively.

Example 12 Impacts of Giant Salvinia Endocides on Other Selected PlantSpecies

General Experimental Procedures:

The experimental species included four herbaceous invasive aquaticspecies of angiosperms (flowering plants), namely, least duckweed (Lemnaminuta) and Brazillan watermeal (Wolffia brasillensis Weddell) of thefamily Araceae, water hyacinth (Eichharnia crassipes) of the familyPontederiaceae and Hydrilla (Hydrilla verticillata (L.f.) Royle) of thefamily Hydrocharitaceae, and two native tree species of gymnosperms,namely baldcypress (Taxodium distichum (L.) Rich.) of the familyCupressaceae and loblolly pine (Pinus taeda L.) of the family Pinaceae.These species are often associated with giant Salvinia in thesoutheastern United States. The experiments were conducted in thegreenhouse (30° C. during the day time and 20° C. at night). Waterextracts were prepared from the dried matter of giant Salvinia (thepreparation method see Example 2). Each species of least duckweed andBrazilian watermeal was separately cultured in nine plastic containers(14×15 cm, 0.68 L) with 400 mL tap water each. The spray experimentstarted until the plants fully covered each of the containers. For eachof these two species, the plants were randomly classified into threegroups: Control: each of the three containers was spayed three timeswith 10 mL of tap water each time; treatment I: each of the threecontainers was spayed twice with 10 mL of 0.1% water extract each timeand then a week later with 10 mL of 5% of water extract; and treatmentII: each of the three containers was spayed twice with 10 mL of 0.5%water extract each time and then a week later with 10 mL of 5% waterextract. Surface coverage of living plants in each container of alltreatments was photographed and measured weekly for six weeks. Fiveplants of each species of water hyacinth and Hydrilla were culturedseparately in container with 50 L tap water. For each species, threecontainers served as control and the plant leaves in each of the otherthree containers were sprayed twice with the total 100 mL of 5% waterextract each time. Plant growth and survival status were documented andphotographed weekly after the treatments for six weeks. Six 2-year oldseedlings of each of bald cypress and loblolly pine in 2-gal pots wereused in the experiment. For each species, three seedlings as controlsreceived water only and the needles and stems in each of the other threeplants were sprayed twice with the total 100 mL of 5% water extract eachtime. Plant growth and survival status were documented and photographedweekly after the treatments for six weeks.

Results:

During the six weeks of experiments, the growth of duckweed andwatermeal had not been inhibited by 0.1%, 0.5% or 5% of water extract ofgiant Salvinia. By the end of the observation, the living plants ofthese three species in all treatments and controls stayed in fullsurface coverage on all containers. Similarly, there was no obvious leafcolor change or damage observed in any treatments of the other twoinvasive flowering plants and two native confer species.

Example 13 Inhibition of Giant Salvinia Plants by Ethanol Extract of theDried Matter of Giant Salvinia

General Experimental Procedures:

Air-dried whole plants (550 g) were ground to a coarse powder andpercolated two times with 95% EtOH at RT (each 3 L, 24 h). The combinedEtOH solution was concentrated to give ethanol extract (34.0 g) underreduced pressure. 1.0 g giant Salvinia ethanol extract was dissolved in2 mL DMSO, and then diluted with ‘water to yield 900 mL 0.1% and 1,000mL 0.01% solutions (g/mL) for further experimental analysis. Theexperiment included 45 healthy and untreated living plants of giantSalvinia (approximately 7 g in fresh weight each). The plants werecultured and tested in the plastic containers (14×15 cm, 0.68 L) in theNCPC Lab at RT. Controls: 15 plants with five in each container (3replications) were cultured with 300 mL of tap water; 0.01% ethanolextract treatment: 15 plants with five in each container (3replications) were sprayed with 300 mL 0.01% of the ethanol extracts ofgiant Salvinia dissolved in tap water; and 0.1% ethanol extracttreatment: 15 plants with five in each container (3 replications) weresprayed with 300 mL 0.1% of the ethanol extracts of giant Salviniadissolved in tap water. Plant growth and survival status were documentedand photographed weekly after the treatments. By the end of 13th week,new growth biomass of plants in each treatment was measured. The biomassof new growth was a primary factor to measure the inhibition of eachtreatment on the target plant.

Results:

By the end of 13 weeks after the treatment, the plants in both Controland 0.01% ethanol extract treatment groups had new growth while about60% of all giant Salvinia plants treated with 0.1% ethanol extract ofthe dried matter of giant Salvinia were dead and no significant growthon the other treated plants although they were still alive.

Example 14 Elimination and Prohibition of Giant Salvinia Plants by WaterExtract of the Dried Matter of Least Duckweed (Lemna minuta)

General Experimental Procedures:

180 g (dry weight) of air-dried whole plants of least duckweed (Lemnaminuta) (Lernnaceae) was ground to a coarse powder and percolated threetimes with 95% EtOH (v/v) at RT (each 2 L, 24 h). After ethanolextraction, the residual plant matters were dried and then percolatedtwo times with water at RT (each 2 L, 24 h). Combined water solution wasconcentrated in vacuo to give water extract (12.2 g). 1.0 g Duckweedwater extract was dissolved in H₂O to yield 900 mL 0.1% and 1,000 mL0.01% solutions (g/mL) for the giant Salvinia inhibition experiment. Thetotal 45 healthy and untreated living plants of giant Salvinia(approximately 7 g in fresh weight each) were cultured and tested inplastic containers (14×15 cm, 0.68 L) in the NCPC Lab at RT. The threetreatments were as follows. Controls: 15 plants with five in eachcontainer (3 replications) were cultured with 300 mL of tap water; 0.01%duckweed water extract treatment: 15 plants with five in each container(3 replications) were sprayed with 300 mL 0.01% of the duckweed extractsdissolved in tap water; and 0.1% duckweed water extract treatment: 15plants with five in each container (3 replications) were sprayed with300 mL 0.1% of the duckweed extracts dissolved in tap water. Plantgrowth and survival status were documented and photographed weekly afterthe treatments. By the end of 13th week, new growth biomass of plants ineach treatment was measured. The biomass of new growth was a primaryfactor to measure the inhibition of each treatment on the target plant.

Results:

At the end of the 13th week of the experiment, each of the 15 giantSalvinia plants in the control group had developed significant newgrowth (FIG. 28). By the same time, in the Treatment I group (0.01%water extract of least duckweed), six giant Salvinia plants haddeveloped significant new growth and four plants had somewhat newgrowth, while the rest five plants were dead and had no new growth. Thetotal new plant growth of the Treatment I was about 40% of that ofControl (FIG. 29). However, all 15 giant Salvinia plants in theTreatment II group (0.1% water extract of least duckweed) were dead andhad no any new growth at all (FIGS. 28 and 29). Further, during theeight months of observation, no recovery or new growth of giant Salviniawere identified thereafter.

Example 15 Elimination and Prohibition of Giant Salvinia Plants byEthanol Extract of the Dried Matter of Least Duckweed

General Experimental Procedures:

Air-dried whole plants (180 g) were ground to a coarse powder andpercolated three times with 95% EtOH (v/v) at RT (each 2 L, 24 h). Thecombined EtOH solution was concentrated under reduced pressure to giveethanol extract (6 g). 1.0 g duckweed ethanol extract was dissolved in 2mL DMSO, and then diluted with water to yield 900 mL 0.1% and 1000 mL0.01% solutions (g/mL) for the inhibition experiment. The total 45healthy and untreated living plants of giant Salvinia (approximately 7 gin fresh weight each) were cultured and tested in plastic containers(14×15 cm, 0.68 L) in the NCPC Lab at RT. The three treatments were asfollows. Controls: 15 plants with five in each container (3replications) were cultured with 300 mL of tap water; 0.01% duckweedethanol extract treatment: 15 plants with five in each container (3replications) were sprayed with 300 mL 0.01% of the duckweed extractsdissolved in tap water; and 0.1% duckweed ethanol extract treatment: 15plants with five in each container (3 replications) were sprayed with300 mL 0.1% of the duckweed extracts dissolved in tap water. Plantgrowth and survival status were documented and photographed weekly afterthe treatments. By the end of 13th week, new growth biomass of plants ineach treatment was measured. The biomass of new growth was a primaryfactor to measure the inhibition of each treatment on the target plant.

Results:

Similar to those observed at the experiment 5, all 15 giant Salviniaplants in the control group had significant new growth at the end of the13th week of the experiment (FIG. 30). By then the total new plantgrowth of the Treatment I group (0.01% ethanol extract of leastduckweed) is less than 15% of that of Control (FIG. 29). During the sametime, 12 giant Salvinia plants in the Treatment II group (0.1% ethanolextract of least duckweed) were dead and had no any new growth at all(FIGS. 29 and 30), and the rest three plants had dead mature tissues butdeveloped somewhat new growth. However, the three new plants did notlast long and eventually all 15 giant Salvinia plants were dead by theend of 15 weeks after the treatment and no new growth thereafter duringthe eight months of observation.

Example 16 Elimination and Prohibition of Floating Fern (SalviniaMinima) Plants by Water Extract of the Dried Matter of Least Duckweed

General Experimental Procedures:

The water extract of least duckweed used in this experiment was the sameas used in Example 6. 1.0 g duckweed water extract was dissolved in H₂Oto yield 900 mL 0.1% and 1,000 mL 0.01% solutions (g/mL) for floatingfern inhibition experiment. The total 90 healthy and untreated livingplants of floating fern Salvinia were cultured and tested in plasticcontainers (14×15 cm, 0.68 L) in the NCPC Lab at RT. The threetreatments were as follows. Controls: 30 plants with 10 in eachcontainer (3 replications) were cultured with 300 mL of tap water; 0.01%duckweed water extract treatment: 30 plants with 10 in each container (3replications) were sprayed with 300 mL 0.01% of the duckweed extractsdissolved in tap water; and 0.1% duckweed extract treatment: 30 plantswith 10 in each container (3 replications) were sprayed with 300 mL 0.1%of the duckweed extracts dissolved in tap water. Plant growth andsurvival status were documented and photographed weekly after thetreatments. By the end of 13th week, new growth biomass of plants ineach treatment was measured. The biomass of new growth was a primaryfactor to measure the inhibition of each treatment on the target plant.

Results:

At the end of the 13th week of the experiment, each of the 30 floatingfern plants in the control group had developed significant new growth(FIG. 31). The floating fern plants had no response with the treatmentat 0.01% water extract of least duckweed during the first 13 weeks ofexperiment (FIGS. 31 and 32). By the same time, the plants treated with0.1% water extract of least duckweed had only about 35% of growth asobserved in the control (FIGS. 31 and 32). However, plants treated with0.01% or 0.1% water extract of least duckweed were all dead by 20 weekswhile untreated plants had new growth.

Example 17 Elimination and Prohibition of Floating Fern (Salviniaminima) Plants by Ethanol Extract of the Dried Matter of Least Duckweed

General Experimental Procedures:

The ethanol extract of least duckweed used in this experiment was thesame as used in the Experiment 7. 1.0 g duckweed ethanol extract wasdissolved in 2 mL DMSO, and then diluted with water to yield 900 mL 0.1%and 1000 mL 0.01% solutions (g/mL) for the inhibition experiment. Thetotal 90 healthy and untreated living plants of floating fern Salviniawere cultured and tested in plastic containers (14×15 cm, 0.68 L) in theNCPC Lab at RT. The three treatments were as follows. Controls: 30plants with 10 in each container (3 replications) were cultured with 300mL of tap water; 0.01% duckweed ethanol 0.01% duckweed extract treatmentextract treatment: 30 plants with 10 in each container (3 replications)were sprayed with 300 mL 0.01% of the duckweed extracts dissolved in tapwater; and 0.1% duckweed ethanol extract treatment: 30 plants with 10 ineach container (3 replications) were sprayed with 300 mL 0.1% of theduckweed extracts dissolved in tap water. Plant growth and survivalstatus were documented and photographed weekly after the treatments. Bythe end of 13th week, new growth biomass of plants in each treatment wasmeasured. The biomass of new growth was a primary factor to measure theinhibition of each treatment on the target plant.

Results:

At the end of the 13th week of the experiment, each of the 30 floatingfern plants in the control group had developed significant new growth(FIG. 33). More than 80% of the floating fern plants were dead after thetreatment at 0.01% ethanol extract of least duckweed and all plantstreated with 0.1% level were dead within the 13 weeks (FIGS. 32 and 33).No new growth was observed thereafter during the eight months ofobservation.

Example 18 Elimination and Prohibition of Brazilian Pepper Tree (Schinusterebinthifolius Raddi) Seedlings by the Ethanol Extract of BrazilianPepper Tree Fruits

General Experimental Procedures:

The fruits of Brazilian pepper tree (S. terebinthifolius) (200 g, in dryweight) and Chinese tallow tree (Triadica sebifera (L.) Small)(Eurphorbiaceae) (260 g, in dry weight) were ground to coarse powdersand extracted two times with 70% ethanol at RT (each 2 L, 48 h). Afterevaporated under reduced pressure, 25.4 g ethanol extract of Brazilianpepper tree and 27 g ethanol extract of Chinese tallow tree (both in dryweight) were obtained, respectively. Each extract was dissolved in waterand then was prepared as experimental solutions at the concentration of10%, respectively. The spray treatment experiment included 36 Brazilianpepper tree seedlings (three weeks old) in pots cultured in thegreenhouse (30° C.). There were six treatments. Control: six seedlingswere sprayed twice with a total 3 mL of tap water each time; surfactanttreatment: six seedlings were sprayed twice with a total 3 mL of tapwater with 7.5 uL Inlet™ and 3.75 uL Kinetin® each time; Brazilianpepper tree extracts treatment: six seedlings were sprayed twice withtotal a 3 mL of 10% pepper tree fruit extracts each time; Brazilianpepper extracts with surfactants treatment: six seedlings were sprayedtwice with a total 3 mL of 10% pepper tree fruit extracts with 7.5 uLInlet™ and 3.75 uL Kinetin® each time; Chinese tallow tree extractstreatment: six seedlings were sprayed twice with total 3 mL of 10%tallow tree fruit extracts each time; and Chinese tallow tree extractswith surfactants treatment: six seedlings were sprayed twice with totala 3 mL of 10% tallow extracts with 7.5 uL Inlet™ and 3.75 uL Kinetin®each time. In addition, newly-spread leaves of six seedlings of each ofthe three native species in North America, namely poison ivy(Toxicodendron radicans (L.) Kuntze or Rhus toxicodendron L.) (familyAnacardiaceae), sweetgum (Liquidambar styraciflua L.) (familyAltingiaceae), and Shumard oak (Quercus shumardii Buckland) (familyFagaceae) were sprayed twice with a total 3 mL of 10% pepper tree fruitextracts each time, respectively. Plant growth and survival status weredocumented and photographed daily after the treatments.

Results:

All Brazilian pepper tree seedlings treated with Brazilian pepper treeextract or the extract with surfactants had significant damages threedays after the first treatments (FIG. 34). Four weeks later, all sixpepper tree seedlings were killed by the pepper tree extract withsurfactants while five seedlings were killed and one was significantlydamaged with dead apical bud and young leaves by the pepper tree extract(FIG. 34). In contrast, the Brazilian pepper tree plants had no damagesin the control, surfactants, 10% ethanol extract of Chinese tallow treefruits, or 10% ethanol extract of Chinese tallow tree fruits withsurfactants treatment. None of the poison ivy, sweetgum, or Shumard oakseedlings had any damages by the Brazilian pepper tree extract.

Example 19 Elimination and Prohibition of Chinese Tallow Tree (Triadicasebifera (L.) Seedlings by the Ethanol Extract of Chinese Tallow TreeFruits

General Experimental Procedures:

The fruits of Chinese tallow tree (Triadica sebifera (L.) Small)(Eurphorbiaceae) (260 g, in dry weight) and Chinese privet (Ligustrumsinense Lour.) (family Oleaceae) (400 g, in dry weight) were ground tocoarse powders and extracted two times with 70% ethanol at RT (each 2 L,48 h), respectively. After evaporated under reduced pressure 27 gethanol extract of Chinese tallow tree fruits and 25.3 g ethanol extractof Chinese privet fruits (both in dry weight) were obtained,respectively. Each extract was dissolved in water and then was preparedas experimental solutions at the concentration of 10%. The spraytreatment experiment included 18 Chinese tallow tree seedlings (threeweeks old) and 12 Chinese privet seedlings under the parent trees in thefield. There were four treatments. Control: six seedlings of eachspecies were sprayed with 3 mL of tap water; Chinese tallow tree extracttreatment on Chinese tallow tree seedlings: six tallow tree seedlingswere sprayed with 3 mL of 10% tallow tree fruit extract; Chinese tallowtree extract treatment on Chinese privet seedlings: six privet seedlingswere sprayed with 3 mL of 10% tallow tree fruit extract; and Chineseprivet extract treatment on Chinese tallow tree seedlings: six tallowtree seedlings were sprayed with 3 mL of 10% Chinese privet fruitextract. Plant growth and survival status were documented andphotographed daily after the treatments.

Results:

All six Chinese tallow tree seedlings treated by the ethanol extract ofChinese tallow tree fruits were significantly damaged (FIG. 35). Duringthe one week of experiments, two tallow tree seedlings were dead andfour were severely injured. However, the tollowtree extract did notcause any damage in Chinese privet seedlings and the Chinese privetextract did not damage the tallow tree seedlings.

Example 20 Elimination and Prohibition of the Red Imported Fire Ant(Solenopsis invicta Buren) by the Fire Ant Extracts and Formic andAcetic Acids

General Experimental Procedures:

Preparation of the fire ant extracts: (1) Acetone extract: 97 g (infresh weight) of the red imported fire ant workers were extracted twotimes with acetone at RT (each 250 mL, 24 h). After evaporated underreduced pressure, the combined extractions yielded 2.13 g (in dryweight) acetone extract. (2) Ethanol extract: 110 g (in fresh weight) ofthe whole worker bodies of the red imported fire ant (Solenopsis invictaBuren) (family Furmicidae) were extracted two times with 95% EtOH at RT(each 250 mL, 24 h). After evaporated under reduced pressure, thecombined extractions yielded 2.72 g (in dry weight) ethanol extract.Both extracts were dissolved in water and then prepared as experimentalsolutions at the concentration of 0.1%, 1%, 5%, and 10%, respectively.Preparation of the formic and acetic acids: Formic acid (88%, SigmaAldrich©) and acetic acid (99.7, ACS reagent grade, VWR InternationalLLC) were prepared as solutions at the concentration of 0.1%, 1%, and 5%immediately before the bioassay experiments. Detection of formic acid byNMR analysis: Approximately 100 fire ant workers were anesthetized byCO₂ and dissolved in 1 mL deuterated CHCl₃ (chroroform). The mixture wasput under ultrasound for 30 min extraction. The fire ants were thenremoved and the extract was dried with anhydrous magnesium sulfate. 500μL of the extract was transferred to NMR tube for detection. ¹H-NMRexperiments were performed on a JEOL ECS 400 spectrometer, withspectroscopic data referenced to the solvent used. According to standardformic acid ¹H-NMR spectrum, the unique singlet of the aldehyde protonshould appear at δ_(H) 8.02. Contact Toxicity Assays: (1) The impacts ofacetone and ethanol extracts of the red imported fire ants on the fireants: the experiment includes nine treatments and each treatment had thetotal 150 workers of the red imported fire ant with 50 ants in each ofthe three 100 mL beakers (as three replications). The ants in eachbeaker were topically sprayed with a total 1 mL of 0.1%, 1%, 5%, or 10%solutions of either acetone or ethanol extract, respectively. Thecontrol group was sprayed with 1 mL pure water. The beakers in alltreatments were covered by cloth to prevent the escape of the fire ants.The surviving number of the fire ants in each treatment was counted by 1h interval for 7 h. (2) The impacts of formic and acetic acids on thefire ants: the experiment had seven treatments and each treatment hadthe total 150 workers of the red imported fire ant with 50 ants in eachof the three 100 mL beakers (as three replications). The ants in eachbeaker were topically sprayed with a total 1 mL of 0.1%, 1%, or 5%solutions of either formic or acetic acids, respectively. The controlgroup was sprayed with 1 mL pure water. The beakers in all treatmentswere covered by cloth to prevent the escape of fire ants. The survivingnumber of the fire ants in each treatment was counted by 1 h intervalfor 7 h. For extracts and acids, LD₅₀ (the dose required to kill half ofthe exposed fire ants) and LD₉₀ (the dose required to kill 90% of theexposed fire ants) were calculated by the PROBIT procedure of SPSS 13.0for Windows. (3) The impacts of combined application of ethanol extractand formic acid on the fire ants: The experiment had the total 720workers of the red imported fire ants with 30 ants in each of the 24Petri Dishes (85 mm in diameter). Control: the ants in three dishes hadno treatment; Water treatment: the ants in each of the three dishes werein contact with 1 mL water for 10 sec; 2.5% ethanol extract treatment:the ants in each of the three dishes were in contacted with 1 mL of 2.5%ethanol extract for 10 sec; 2.5% formic acid treatment: the ants in eachof the three dishes were in contacted with 1 mL of 2.5% formic acid for10 sec; mixture of ethanol extract (2.5%) and formic acid (2.5%)treatment: the ants in each of the three dishes were in contacted with 1mL of a mixture of ethanol extract and formic acid (each had 2.5% inconcentration in the mixture) for 10 sec; 5% ethanol extract treatment:the ants in each of the three dishes were in contacted with 1 mL of 5%ethanol extract for 10 sec; 5% formic acid treatment: the ants in eachof the three dishes were in contacted with 1 mL of 5% formic acid for 10sec; and mixture of ethanol extract (5%) and formic acid (5%) treatment:the ants in each of the three dishes were in contacted with 1 mL of amixture of ethanol extract and formic acid (each had 5% in concentrationin the mixture) for 10 sec. The surviving number of the fire ants ineach treatment was counted by 15 min interval for 90 min. (4) Theimpacts of combined application of ethanol extract and formic acid onthe injured fire ants: The 450 workers of the red imported fire antswere placed in 15 Petri Dishes (85 mm in diameter) with 30 in each dish.The ants were anesthetized by CO₂ and then each ant was injured in itsgaster by pin. Control: the ants in three dishes had no treatment; Watertreatment: the ants in each of the three dishes were in contact with 1mL water for 10 sec; 1.25% ethanol extract treatment: the ants in eachof the three dishes were in contacted with 1 mL of 1.25% ethanol extractfor 10 sec; 1.25% formic acid treatment: the ants in each of the threedishes were in contact with 1 mL of 1.25% formic acid for 10 sec; andmixture of ethanol extract (1.25%) and formic acid (1.25%) treatment:the ants in each of the three dishes were in contacted with 1 mL of amixture of ethanol extract and formic acid (each had 1.25% inconcentration in the mixture) for 10 sec. The surviving number of thefire ants in each treatment was counted by 15 min interval for 90 min.(5) The impacts of ethanol extract of the red imported fire ants onsubterranean termite (Reticulitermes flavipes (Kollar)) (familyRhinotermitidae): the experiment included five treatments and eachtreatment had 150 subterranean termites with 20 workers, 10 soldiers and5 winged reproductive ants in each of the three 100 mL beakers (as threereplications). The termites in each beaker were topically sprayed with atotal 1 mL of 0.1%, 1%, 5%, or 10% solutions of ethanol extract,respectively. The control group was sprayed with 1 mL pure water. Thebeakers in all treatments were covered by cloth to prevent the escape offire ants. The surviving number of the fire ants in each treatment wascounted by 1 h interval for 7 h. Fumigation Toxicity Assays of FormicAcid: The experiment included approximately 300 workers of the fire antsin each container with the filter paper treated with 10 mL of 0.1%, 1%,or 5% formic acid, respectively. The surviving number of the fire antsin each treatment was counted by 1 h interval for 2 h. LD₅₀ (the doserequired to kill half of the exposed fire ants) and LD₉₀ (the doserequired to kill 90% of the exposed fire ants) were calculated by thePROBIT procedure of SPSS 13.0 for Windows.

Results:

The topical applications of both the extracts of the red imported fireants and the organic acids inhibited the fire ants. Both formic acid andacetic acid were not detected in chloroform, acetone, or ethanol extractaccording to the NMR analysis. The ethanol extract showed moresignificant toxicity against the fire ants than the acetone extract.During the 7 h contact toxicity bioassays, an average of approximately60%, 70%, or 80% of the fire ants in contact with 1%, 5%, or 10% ethanolextract were dead, respectively (FIG. 36). In contact toxicitybioassays, 7 h LD₅₀ and LD₉₀ of formic acid, acetic acid, ethanolextract, and acetone extract for the fire ants was 1.9% and 5.09%, 6.42%and 9.96%, 1.81% and 15.58%, and 18.67% and 34.18%, respectively. Formicacid had a more significant impact on survival of fire ants than aceticacid. During the 90 min of contact toxicity bioassay, the averagemortality with 2.5% and 5% formic acid was more than 70% or 100%,respectively (FIG. 37). In fumigation bioassays, 2 h LD₅₀ and LD₉₀ offormic acid for the fire ants was 0.5% and 0.9%, respectively. Thecombined application of ethanol extract of the fire ants and formic acidhad more significant effects on fire ant survival than use of eitherethanol extract or formic acid alone (FIGS. 37 and 38). However, theethanol extract of the fire ants had no impacts on subterranean termite.

Example 21 Elimination and Prohibition of the Subterranean Termite(Reticulitermes flavipes (Kollar)) by Formic Acid

General Experimental Procedures:

Formic acid (90.4%, certified ACS reagent grade, Fisher ScientificCompany, Fair Lawn, N.J., USA) was prepared as solutions at theconcentration of 0.1%, 1%, and 5% immediately before the bioassayexperiments. Detection of formic acid by NMR analysis: Approximately 100termites were anesthetized by CO₂ and dissolved in 1 mL deuterated CHCl₃(chroroform). The mixture was put under ultrasound for 30 minextraction. The termites were then removed and the extract was driedwith anhydrous magnesium sulfate. 500 μL of the extract was transferredto NMR tube for detection. ¹H-NMR experiments were performed on a JEOLECS 400 spectrometer, with spectroscopic data referenced to the solventused. According to standard formic acid ¹H-NMR spectrum, the uniquesinglet of the aldehyde proton should appear at δ_(H) 8.02. ContactToxicity Assays: The experiment included five treatments and eachtreatment had total 105 subterranean termite (Reticulitermes flavipes(Kollar)) (family Rhinotermitidae) including 20 workers, 10 soldiers and5 winged reproductives (alates) in each of the three 100 mL beakers (asthree replications). The termites in each beaker were topically sprayedwith a total 1 mL of 0.1%, 1%, 5%, or 10% solutions of formic acid,respectively. The control group was sprayed with 1 mL pure water. Thebeakers in all treatments were covered by cloth to prevent the escape oftermites. The surviving number of the termites in each treatment wascounted by 1 h interval for 7 h.

Results:

The NMR analysis indicated that the subterranean termites containedsignificant amount of formic acid (FIG. 39). Formic acid at higherconcentrations (5 or 10%) killed all termites (including workers,soldiers, and reproductive) during the first hour of contact experiment.During the 7 h of contact toxicity bioassays, formic acid at 1%concentration killed more than 40% of the termites (FIG. 40). However,formic acid at 0.1% concentration had no impact on the termites. In thecontact toxicity bioassays, 7 h LD₅₀ and LD₉₀ of formic acid for thetermite was 4.71% and 9.08%, respectively.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents that are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method of controlling a first invasivespecies comprising applying a composition comprising a natural pesticideto the first invasive species, wherein the natural pesticide is derivedand/or isolated from Triadica sebifera, wherein the first invasivespecies is the same as or is within the same family as Triadicasebifera, and wherein the natural pesticide consists of an extract ofTriadica sebifera.
 2. The method of claim 1, wherein the growth,reproduction, and/or spread of the first invasive species is haltedwithin 1 week or 1 month and/or is halted for at least 1 year.
 3. Themethod of claim 1, wherein the composition comprising the naturalpesticide is applied topically to the first invasive species; sprayedonto the first invasive species, spread around the first invasivespecies; and/or dissolved in water surrounding the first invasivespecies.
 4. The method of claim 1, wherein the natural pesticideconsists of an ethanol extract of Triadica sebifera or fraction thereof.5. The method of claim 1, wherein the first invasive species is Triadicasebifera.
 6. The method of claim 5, wherein the natural pesticideconsists of an ethanol extract of Triadica sebifera or fraction thereof.